the response to heat shock and oxidative stress ... · e-mail: [email protected]...

YEASTBOOK

CELL SIGNALING & DEVELOPMENT

The Response to Heat Shock and Oxidative Stressin Saccharomyces cerevisiaeKevin A. Morano,* Chris M. Grant,† and W. Scott Moye-Rowley‡,1

*Department of Microbiology and Molecular Genetics, University of Texas Medical School and Graduate School of Biomedical Sciences, Houston, Texas77030, †Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom, and ‡Department of Molecular Physiology andBiophysics, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242

ABSTRACT A common need for microbial cells is the ability to respond to potentially toxic environmental insults. Here we review theprogress in understanding the response of the yeast Saccharomyces cerevisiae to two important environmental stresses: heat shockand oxidative stress. Both of these stresses are fundamental challenges that microbes of all types will experience. The study of theseenvironmental stress responses in S. cerevisiae has illuminated many of the features now viewed as central to our understanding ofeukaryotic cell biology. Transcriptional activation plays an important role in driving the multifaceted reaction to elevated temperatureand levels of reactive oxygen species. Advances provided by the development of whole genome analyses have led to an appreciation ofthe global reorganization of gene expression and its integration between different stress regimens. While the precise nature of thesignal eliciting the heat shock response remains elusive, recent progress in the understanding of induction of the oxidative stressresponse is summarized here. Although these stress conditions represent ancient challenges to S. cerevisiae and other microbes, muchremains to be learned about the mechanisms dedicated to dealing with these environmental parameters.

TABLE OF CONTENTS

Abstract 1157

Introduction 1158

Control and Regulation of the Heat Shock Response 1158The major players: heat shock transcription factor 1 and Msn2/4 1159

HSF1: 1159Msn2/4: 1160Sensing thermal stress: 1160

Modulators of the HSR 1161Chromatin modulation of heat shock gene expression: 1161Effects of heat shock on RNA metabolism: 1162Modulation by trans-acting factors: 1162

Global analysis of the HSR 1162

Cellular functions of the heat shock response and heat shock proteins 1163The yeast “chaperome”: 1163

Continued

Copyright © 2012 by the Genetics Society of Americadoi: 10.1534/genetics.111.128033Manuscript received February 21, 2011; accepted for publication April 29, 20111Corresponding author: Department of Molecular Physiology and Biophysics, Carver College of Medicine, 6-530 Bowen Science Bldg., University of Iowa, Iowa City, IA 52242.E-mail: [email protected]

Genetics, Vol. 190, 1157–1195 April 2012 1157

mailto:[email protected]

CONTENTS, continued

The Hsp70 chaperone system: 1163The Hsp90 chaperone system: 1165Oligomeric HSPs: Hsp104 and the small HSPs: 1166Chaperone networks: 1166

The intersection between the HSR and other stress responses 1167HSR and oxidative stress response: 1167HSR and cell wall integrity: 1167

Yeast as a model system for understanding the HSR 1168Exploiting yeast to understand the human HSR: 1168The HSR in other yeasts: 1168

The Oxidative Stress Response 1168Sources of ROS generation in yeast cells 1169

Commonly used model ROS compounds 1169Hydroperoxides: 1169The superoxide anion: 1170Thiol-reactive compounds: 1170Heavy metal stress: 1170

Transcriptional control of the OSR 1170Yap1: 1170Nuclear localization of Yap1 responds to oxidative stress: 1170Yap1 homologs and redox stress: 1172Skn7: 1172Msn2/4: 1174

Antioxidant defenses 1175Catalases: 1175Superoxide dismutases: 1175Methionine sulfoxide reductase: 1175Thioredoxins: 1175Peroxiredoxins: 1177The glutathione system: 1177Glutaredoxins: 1178Glutathione peroxidases: 1179Glutathione transferases: 1179Ascorbic acid: 1179

Cellular responses to ROS 1179Redox homeostasis and oxidative stress: 1180Translational regulation of gene expression: 1181Metabolic reconfiguration is a rapid, regulated response to oxidative stress: 1182Control of the cell division cycle vs. apoptotic cell death following oxidative stress: 1183

Future directions 1184

ORGANISMS are constantly challenged by ever-changingvariables in their environment, including fluctuating

nutrient levels, osmotic imbalance, exposure to toxic mole-cules, and nonoptimal temperatures. While multicellular ormotile organisms can usually alter these conditions bya change in location or physiology, single-celled organismssuch as yeast are at the mercy of their situation, and mustadapt or perish. Even in the absence of lethal stresses, subtledifferences in the ability to tolerate environmental fluctua-tions can confer a competitive advantage in a mixed nichesuch as the surface of a ripening fruit. Here we will reviewthe current state of knowledge of the response of Saccharo-

myces cerevisiae to two important environmental challenges:heat and oxidative stress. Studies on the response to heatand oxidative stress have provided important insight intonearly every aspect of eukaryotic cell biology, as we hopewill be clear from our discussion.

Control and Regulation of the Heat Shock Response

The most fundamental stress experienced by a yeast cell isthe ambient temperature. Saccharomyces cerevisiae exhibitsoptimal growth between 25� and 30� (77�–86�F), the an-thropomorphic equivalent of a sunny day. However, at

1158 K. A. Morano, C. M. Grant, and W. S. Moye-Rowley

temperatures .36–37� (nearing 100�F), yeast cells activatea protective transcriptional program termed the heat shockresponse (HSR) and alter other components of their physiol-ogy, including membrane composition and carbohydrate flux.S. cerevisiae and other mesophilic yeasts maintain growth attemperatures up to �42� (107�F), but are unable to cope withchronic exposure to higher temperatures (indeed, yeast RNApolymerase II is inactive at temperatures .42�; Yamamotoet al. 2008). As yeast cells may experience this range of tem-perature over the course of a day/night cycle, investigation ofthe HSR induced by shift from 30� to 37� (the classic “heatshock”) in a laboratory setting is physiologically relevant. Overthe last 25 years, much has been revealed about how yeastcells respond to heat shock, including the transcription factorsthat govern changes in gene expression and the metabolicreprogramming that enables cells to withstand chronic expo-sure to sublethal temperatures. In the decade since the pre-vious incarnation of this review series, the scope has been bothnarrowed, by detailed analysis of molecular interactions be-tween players, and expanded, through genome-level surveysof gene expression and protein–protein interactions. Theserecent advances will be discussed in this chapter, with anemphasis on the integration and interplay between the HSRand other stress response pathways. Because the literature isextensive, the reader is directed to several prior reviews thatdiscuss the relevance of past findings in greater depth (Piper1997; Trott and Morano 2003).

The major players: heat shock transcription factor 1and Msn2/4

In eukaryotes, the heat shock transcription factor (HSF)protein family is the primary modulator of the HSR. In S.

cerevisiae (and likely other related yeasts), a second transcrip-tion factor represented by the MSN2 and MSN4 genes alsocontributes substantially to heat shock gene expression. In-deed, microarray analysis using conditional and knockoutmutants, respectively, of the HSF1, MSN2, and MSN4 genessuggests that these three factors are responsible for the bulk ifnot the entirety of the HSR (Figure 1). The Msn2/4 regulon ismuch broader than heat shock-induced transcripts andincludes oxidative stress and metabolic and other cytoprotec-tive genes, leading to its characterization as the “environmen-tal stress response” (ESR), described in more detail below.

HSF1: Four distinct HSF isoforms have been described inmammals. HSF1 is primarily dedicated to regulating theHSR, while HSF2 plays a role in developmental geneexpression. The roles of HSF3 and HSF4 are less wellunderstood but evidence suggests these factors may func-tionally interact with HSF1 to modulate gene expression(reviewed in Akerfelt et al. 2010). Invertebrates includingthe “lower” eukaryotes express a single, essential HSF equiv-alent to mammalian HSF1 (Akerfelt et al. 2010). The fun-damental architectural elements of this transcription factorare conserved, consisting of an amino-terminal winged he-lix-turn-helix DNA binding domain (DBD) followed by a leu-cine zipper motif required for trimerization and activation,a loosely defined, serine-rich “regulatory” domain, and acarboxy-terminal transcriptional activation domain (Trottand Morano 2003). S. cerevisiae HSF1 is unusual in that itincludes an amino-terminal extension of �150 amino acidsthat acts as a second potent transcriptional activation do-main (Sorger 1990). Hsf1 recognizes a pentameric heatshock element (HSE) defined as repeating units of the se-quence nGAAn (Sorger and Pelham 1987). Early investiga-tions defined the HSE as a triple inverted repeat, consistentwith the fact that Hsf1 binds DNA as a trimer (Sorger andPelham 1987). The discovery that Hsf1 activates the yeastmetallothionein gene CUP1 in response to heat shock andoxidative stress challenged this paradigm, as the CUP1 pro-moter contains an atypical HSE with two nGAAn repeatsfollowed by a pentameric spacer and another repeat (Tamaiet al. 1994; Liu and Thiele 1996) (Figure 2). Subsequentwork revealed that variations of this “noncanonical” HSEwere widespread in the yeast genome (Yamamoto et al.2005), greatly expanding the range of possible Hsf1 targets(see Figure 2).

Figure 1 Regulation of Msn2/4 and Hsf1. Regulation of Msn2 by growthcontrol proteins and oxidative stress is diagrammed. H2O2 triggers theoxidation of cytoplasmic thioredoxin proteins (Trxox) from their normallyreduced status (Trxred). This induces the recruitment of Msn2 into thenucleus where it can interact with its cognate binding site (stress responseelement, STRE) and activate expression of target gene transcription. Hsf1is constitutively nuclear and prebound to many target genes containingheat shock elements (HSEs) in their promoters. Hsf1 is also regulated bythe nutrient-sensing kinases, Snf1 and PKA, and by oxidative stressthrough unknown mechanisms.

Figure 2 Divergence in HSE architecture. Three different types of HSEhave been described on the basis of spacing and positioning, as describedin the text. Many genes within the Hsf1 regulon have annotated HSEswithin the promoters; representative examples are shown (Yamamotoet al. 2005). n, any nucleotide.

Heat and Oxidant Resistance 1159

http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000004640














HSE architecture dramatically dictates Hsf1 behavior, asthe noncanonical HSE with 5-bp “spacers” between thenGAAn units is cooperatively bound by HSF that need notbe phosphorylated nor oligomerized (trimerized) (Hashikawaet al. 2006), two conditions previously thought to be abso-lute prerequisites for function. Moreover, a novel C-terminalregulatory domain (CTM) identified in Hsf1 is required fortranscription from “perfect” promoters bound noncoopera-tively by a single trimer, but not for the noncanonical genetargets (Hashikawa et al. 2006). These findings demonstratepreviously unappreciated complexity in Hsf1 function in S.cerevisiae and help explain the unexpectedly large number ofHsf1-responsive genes uncovered in genomic analyses (seeGlobal analysis of the HSR, below).

Msn2/4: Msn2 and its close homolog Msn4 (referred to asMsn2/4) were identified in the mid-1990s as proteins re-quired for expression of a wide array of genes in responseto multiple types of stress, but not for viability under normalconditions (Martinez-Pastor et al. 1996; Schmitt andMcEntee 1996). Analysis of the promoter of the cytosoliccatalase gene (CTT1) indicated the presence of an elementtermed the stress responsive element (STRE) that consistedof a pentameric core of CCCCT (Wieser et al. 1991). TheSTRE was demonstrated to be the recognition site for Msn2/4 and to be required for induction of CTT1 and many othergenes to various stresses (Martinez-Pastor et al. 1996). Msn2is the dominant factor of the two, although overexpressionof MSN4 can partially alleviate gene expression and growthphenotypes associated with msn2Δ cells (Schmitt andMcEntee 1996). Msn2/4 targets are regulated by growthphase and nutrient status (Figure 1); i.e., induced after thediauxic shift or in late stationary/quiescent phase cells(Trott and Morano 2003). Nutrient-responsive transcrip-tional control is mediated by the glucose-responsive cyclicAMP (cAMP)-protein kinase A signaling pathway that phos-phorylates Msn2/4 under nonstress conditions to block itsnuclear import, thereby inhibiting target gene activation(Gorner et al. 1998). While PKA is an important negativeregulator of Msn2/4 function, other signal transductionpathways modulate the activity of these transcription fac-tors. The AMP-dependent protein kinase Snf1 also phos-phorylates and downregulates Msn2 (De Wever et al.2005). Experiments from several labs have implicated theprotein phosphatase PP1 in dephosphorylation of the PKA-and Snf1-triggered changes in phosphorylation (Mayordomoet al. 2002; De Wever et al. 2005; Lenssen et al. 2005). PP1appears to directly dephosphorylate Msn2 but also to nega-tively influence Snf1 activity, thus providing two differentroutes of control to Msn2 activity. Utilization of these mul-tiple distinct but related pathways to control Msn2/4 func-tion is likely to provide the means through which Msn2regulation is tied into general cellular stress.

Nucleocytoplasmic shuttling of Msn2/4 is rapid and oscil-lates even during stress conditions, suggesting that modula-tors such as cAMP/PKA influence the sensitivity and

frequency of nuclear localization rather than absolutely re-stricting transport from one compartment to another (Jacquetet al. 2003). Msn2 is degraded in the nucleus upon consti-tutive or chronic activation, providing a potential adaptationmechanism for periods of prolonged nutrient limitation orenvironmental stress (Lallet et al. 2004). Remarkably, elim-ination of Msn2/4 allows cells to dispense with the “essen-tial” cAMP/PKA pathway, suggesting that one or moreMsn2/4 targets antagonize PKA function and are detrimen-tal to optimal growth (Smith et al. 1998). Several lines ofevidence point to the Yak1 kinase as the prime candidate forthis role. First, Msn2/4 controls YAK1 expression (Smithet al. 1998); second, Yak1 kinase activity is directly inhibitedby phosphorylation by PKA (Lee et al. 2008); and third, de-letion of YAK1 also suppresses PKA hypomorphic mutations(Garrett and Broach 1989). In addition, Yak1 phosphory-lates the PKA regulatory subunit, Bcy1, biasing its localiza-tion to the cytoplasm. In contrast to the negative regulationexerted on Msn2/4 by PKA, Yak1 phosphorylates andactivates Msn2/4 and Hsf1 under low glucose conditions(Griffioen et al. 2001; Lee et al. 2008). This finding makesthis kinase one of the few regulatory proteins known to in-fluence both Hsf1 and Msn2/4 (Griffioen et al. 2001; Lalletet al. 2004; Lee et al. 2008).

Sensing thermal stress: How do yeast cells sense heat shockand activate the appropriate transcriptional response? In1991 Craig and Gross postulated that heat shock proteins(HSPs) could act as the cellular thermometer, but thisconjecture lacked substantial supporting evidence in eukary-otic systems (Craig and Gross 1991). Moreover, this idea ispredicated on the fact that HSPs are sensing a physiologicaldisturbance distinct from those caused by other stresses. In-sight into this question is provided by the observation thatforced misfolding of nascent proteins by incorporation of theproline analog azetidine 2-carboxylic acid (AZC) results inG1 arrest in yeast cells in a manner similar to heat shock(Rowley et al. 1993; Trotter et al. 2001). AZC treatment alsoresults in transcriptional activation of a set of genes closelymatching the Hsf1, but not the Msn2/4 regulon, and repres-sion of ribosomal protein synthesis, an Hsf1-dependent pro-cess (Trotter et al. 2002). Treatment with sublethalconcentrations of ethanol (6–8%) also induces Hsf1 butnot Msn2/4 (Takemori et al. 2006). Lastly, inhibition of pro-teasomal degradation with the specific inhibitor MG132 isalso an effective activator of Hsf1 (Lee and Goldberg 1998).Together these data suggest that accumulation of misfoldedproteins triggers Hsf1 activation, but it is not clear whetherit is the accumulation of misfolded forms of existing pro-teins, nascent chains, or both that is the proximal inducer.While cycloheximide pretreatment predictably blocks Hsf1induction by AZC incorporation, the same experiment hasnot been done with ethanol (Trotter et al. 2002). Both treat-ments, as well as transient heat shock, fail to induce theunfolded protein response (UPR) in the ER, which is stimu-lated by misfolding of resident proteins (Cox and Walter




































































1996). Moreover, heat shock at 37� does not result in bulkprotein aggregation (Nathan et al. 1997). These findingssupport a model whereby misfolding of newly synthesizedpolypeptides might be sensed as increased substrate load onribosome-proximal HSPs/chaperones. How is this signalthen transmitted to Hsf1? Although a small number ofHsf1 post-translational regulators have been uncovered(see below), substantial genetic data in yeast supporta model wherein select HSPs, including Hsp70, Hsp90,and their cofactors, repress Hsf1 activation (Nelson et al.1992; Duina et al. 1998; Liu et al. 1999; Harris et al.2001). Moreover, pharmacological inhibition of Hsp90 withspecific small molecules such as the ansamycins (geldana-mycin and macbecin) or radicicol results in Hsf1 activationin yeast, as it does in higher eukaryotes (Zou et al. 1998;Harris et al. 2001). Given the high protein synthetic capacityof actively growing cells, it is conceivable that a transientheat shock could impact the folding of enough nascentchains or newly released polypeptides to effectively competefor components of the Hsp70/Hsp90 machinery with Hsf1.However, yeast Hsf1 is largely trimerized, nuclear, and DNAbound in unstressed cells (McDaniel et al. 1989; Hahn et al.2004); therefore, the chaperone repression model has to berevised: protein misfolding (in the nucleus) must be sensedby nuclear chaperones or cytoplasmic misfolding eventsmust titrate chaperones that transit between the two com-partments, depleting the nuclear pool. In support of thislatter argument, both Hsp70 (Ssa4) and Hsp90 (Hsp82and Hsc82) can localize to the nucleus in response to envi-ronmental stress (Chughtai et al. 2001; Quan et al. 2004;Tapia and Morano 2010).

In addition to the Hsf1 pathway, thermal stress also indu-ces the Msn2/4 regulon and activates the cell wall integritypathway. Although the mechanism of Msn2/4 activation bylow glucose conditions through cAMP/PKA is well estab-lished, induction of this pathway by heat shock and otherenvironmental stress is poorly understood (Thevelein et al.2000). Like Hsf1, Msn2/4 is hyperphosphorylated in re-sponse to heat shock, but this modification is inhibited bycAMP, suggesting that it is not mediated by PKA (Garreauet al. 2000). Heat shock, oxidative and ethanol stress mod-erately reduce cAMP levels via destabilization of the Rasactivator, Cdc25, leading to speculation that cAMP/PKAmay yet be the nexus of ESR signaling and that additionalphosphorylation events may act as subordinate modulatorsof the response (Wang et al. 2004). Activation of the cellwall integrity pathway by heat shock has been reviewedelsewhere, but importantly is insulated from Hsf1 orMsn2/4 activation and is not affected by cAMP/PKA (Levin2005).

Modulators of the HSR

Chromatin modulation of heat shock gene expression:Because most of the HSR is by definition a transcriptionalprogram, it is not surprising that chromatin status plays an

important role in governing the magnitude and kinetics ofthe response. It is generally accepted that transcriptionallyinert portions of eukaryotic genomes are maintained indensely packed, nucleosome-rich states. Moreover, associa-tion of the DNA with nucleosomal proteins is enhanced bydeacetylation of lysine residues in the histone tails, as wellas other epigenetic modifications, including methylation.The HSP82 locus, one of two genes encoding the chaperoneHsp90, has been an informative model for how heat shockrapidly and reversibly activates gene expression. EarlyDNase I footprinting studies established that the HSP82 pro-moter contains multiple HSEs, only one of which (HSE1)was constitutively bound by Hsf1 in unstressed cells (Grosset al. 1990). Mutation of HSE1 resulted in the appearance oftwo stable nucleosomes, suggesting that Hsf1 binding “pre-cleared” the promoter (including the TATA box), priming itfor transcription initiation (Gross et al. 1993). Upon heatshock, Hsf1 occupies additional low-affinity HSEs withinthe HSP82 promoter in a cooperative manner, which cancompensate for loss of HSE1 with regard to both nucleo-some clearance and gene expression (Erkine et al. 1999).This mode of Hsf1 activation may be the norm, as ChIP-chipstudies demonstrated that Hsf1 binds to many target genes(see below) constitutively, with additional binding occurringin response to heat shock (Hahn et al. 2004). Strikingly,nucleosome remodeling kinetics are exceedingly rapid—abundance of histone 4 within the HSP82 locus is drasticallyreduced within 1 min of heat shock (Zhao et al. 2005a).Hsf1 target genes attenuate expression within 20–40 minafter induction, and correspondingly, nucleosomes are foundto reposition within the promoter, ORF, and 39-UTR ofHSP82 in the same timeframe (Zhao et al. 2005a). The cor-relation between heat shock gene induction and nucleosomeremodeling may not be absolute; the remodeling complexSWI/SNF is recruited to Hsf1 target genes and required formaximal gene expression, yet histone eviction occurs insnf2Δ mutants that abrogate complex function, albeit withdelayed kinetics (Shivaswamy and Iyer 2008).

A genome-wide search for genes required for sustainedgrowth during heat shock (39�) resulted in the identificationof multiple components of the Rpd3L deacetylase complex(RPD3, SIN3, UME1, SAP30, SDS3, DEP1, and PHO23)(Ruiz-Roig et al. 2010). Rpd3L promotes transcription initi-ation at target promoters, where it functions in both activat-ing and repressing contexts. Intriguingly, the complex isrequired for activation of Msn2/4-dependent gene expres-sion in response to heat shock, where it contributes to themagnitude of the response; i.e., genes are induced by heatshock but to a much lower level than in wild-type strains(Alejandro-Osorio et al. 2009). In contrast, Hsf1-dependentgenes such as HSP82 are activated independently of Rpd3L,which instead contributes to basal repression (Kremer andGross 2009; Ruiz-Roig et al. 2010). Rpd3L is recruited topromoters upon stress, and it likely contributes to subse-quent recruitment of Msn2 (Ruiz-Roig et al. 2010). Chroma-tin modification therefore plays a critical role in modulating
















































the kinetics and amplitude of stress gene expression, sug-gesting close collaboration between stress-specific transcrip-tion factors and more general gene control machinery.

Effects of heat shock on RNA metabolism: In addition tomodulation of gene transcription, heat shock causes imme-diate post-transcriptional effects with significant physiolog-ical impact. Remarkably, upon 42� heat shock bulk poly(A)+

RNA stably accumulates in the nucleus (Saavedra et al.1996). This finding is at odds with the obvious robust trans-lation of HSPs under the same conditions. In fact, heat shocktranscripts, best exemplified by studies with the SSA4 gene,are selectively exported through nuclear pores utilizing sig-nals in the 59- and 39-untranslated regions (UTRs) of themessage (Saavedra et al. 1996). A specific nuclear pore pro-tein Rip1 is required for export of heat shock transcriptsduring thermal stress but not under normal growth condi-tions, defining a dedicated transport pathway for these im-portant mRNAs (Saavedra et al. 1997). Recent work hasdefined novel ribonucleoprotein assemblies termed process-ing bodies (P-bodies) and stress granules (SGs) that appearto concentrate nontranslating mRNAs in exchangeable butsequestered pools in response to a variety of stress condi-tions (Parker and Sheth 2007). Heat shock induces the for-mation of SGs that contain translation initiation factors andnonheat shock mRNAs (for example, PGK1) capable of redis-tributing into the cytoplasm, and presumably reengaging intranslation, upon recovery (Grousl et al. 2009). Heat shock-induced SGs also contain a subset of P-body componentsinvolved in RNA degradation including Dcp2 and Dhh1,yet are spatially distinct from other P-body markers (Grouslet al. 2009). These data demonstrate that cells restrict thetranslation of nonheat shock transcripts via at least twomechanisms: blocking mRNA transport from the nucleusand redirecting cytoplasmic mRNAs away from ribosomesand into subcellular complexes. Interestingly treatment ofyeast cells with high ethanol concentrations (.10%) alsoleads to block of mRNA export and formation of SGs(Saavedra et al. 1996; Kato et al. 2011). It is tempting tospeculate that this result may be yet another manifestationof the close relationship heat and ethanol share as proteindenaturants that lead to activation of the HSR, but a plausi-ble mechanism to account for this has not been put forward.In addition, the possibility that both treatments similarlyaffect other aspects of yeast physiology such as membranefluidity cannot be excluded.

Modulation by trans-acting factors: Great progress hasbeen made in understanding regulation of HSF1 in humanand murine systems by post-translational modifications,including phosphorylation by multiple kinases and mostrecently reversible acetylation mediated by sirtuin deacety-lase enzymes (Westerheide et al. 2009; Akerfelt et al. 2010).In contrast, relatively little is known in this regard in yeast.As described earlier, Msn2/4 is under tight control by thecAMP/PKA pathway (mediated in part by Yak1), and target

serine residues phosphorylated by PKA have been identifiedin Msn2 whose mutation abrogates nutrient control of func-tion (Gorner et al. 2002). Similarly, Hsf1 activates a subsetof genes, including the copper metallothionein CUP1 in re-sponse to low glucose conditions, and induction is lost incells lacking the AMP kinase homolog Snf1 (Tamai et al.1994). Snf1 was found to directly phosphorylate Hsf1 inresponse to glucose starvation, but not in response to heatshock, demonstrating that distinct pathways sense environ-mental changes and communicate this information to Hsf1(Hahn and Thiele 2004). Hsf1 phosphorylation patterns aredifferent in response to low sugar, heat shock, or oxidativestress, but kinases mediating phosphorylation in the lattertwo cases are yet to be discovered (Tamai et al. 1994; Liuand Thiele 1996; Hahn and Thiele 2004). It is not clear atthis time whether such kinases would themselves be acti-vated by the specific stress, as is Snf1, or if constitutivelyactive kinases gain access to Hsf1 under stress conditions.

Global analysis of the HSR

As is the case for analysis of many transcriptional networksin yeast, the advent of DNA microarray technology offersunprecedented insight into the breadth of the heat shockresponse on a genome-wide scale. Two comprehensivestudies utilized microarray approaches to reveal changes ingene expression in response to stressful conditions rangingfrom heat shock to osmotic stress to nutrient limitation.Gene expression of �10% of the genome is remodeled dur-ing one or more stresses, highlighting the depth to whichstress-based transcription penetrates the global transcrip-tome (Gasch et al. 2000; Causton et al. 2001). Abundance(probably reflecting transcriptional changes but could alsoinclude differences in mRNA stability (Castells-Roca et al.2011) of �600 genes was found to decrease during stress,while about half that number (300) are induced (Gaschet al. 2000; Causton et al. 2001). This comprehensive re-sponse has been termed the environmental stress response(ESR). Comparative arrays using an msn2Δ msn4Δ mutantdemonstrated that a significant proportion of the inducedgenes rely on these transcription factors for activation(Gasch et al. 2000). Notably, many of these same targetsare activated in response to multiple stresses, consistentwith previous single-gene analyses. The HSR is thereforea subset of the ESR and is composed of genes requiringHsf1, Msn2/4, both, or in a few isolated cases, neither fortheir expression during heat shock. Subsequent studies toidentify direct Hsf1 targets cataloged �72 genes whose heatshock induction at 39� was abrogated by a severe loss-of-function mutant, hsf1-R206S/F256S (Yamamoto et al. 2008).ChIP analysis to identify promoters directly bound by Hsf1revealed a larger number (165) of potential Hsf1-responsivegenes (Hahn et al. 2004). This result suggests that Hsf1 maycontribute to the expression of nonheat shock genes and isconsistent with the fact that it has been previously docu-mented to bind many promoters constitutively. An alternativeexplanation may be that the “normal” conditions established






























in the laboratory (rich medium, high glucose, aeration, 30�)may actually be perceived as a mild stress by yeast cells.While this interpretation would make the behavior of yeastHsf1 more like its mammalian counterpart, a closer analogymay be Drosophila HSF1, which is also constitutively nuclearand prebound to many heat shock promoters (Yao et al.2006).

A number of additional insights can be gleaned fromthese genome-wide studies. It is clear that the magnitude ofthe HSR is proportionate to the intensity of the stress:temperature shift from 25� to 37� results in a longer lastingHSR with greater amplitude of change in gene expressionwhen compared to a 29�-to-33� shift (Gasch et al. 2000).There is a limit to this effect, as no significant differences inbinding of Hsf1 to chromosomal loci are detected between39� and 42� (Hahn et al. 2004). These data suggest thatyeast cells are capable of sensing gradations in temperaturestress up to a threshold point, after which the system, atleast Hsf1, is maximally activated. A handful of Hsf1 targetgenes (CUP1 and HSP82) are induced more strongly at 39�vs. 37�, demonstrating that the threshold is likely in the 39�–40� range (Santoro et al. 1998).

Another important insight is the breadth of target genesinduced by the HSR. While protein chaperones and theircofactors are obvious and for the most part long-recognizedtargets, the HSR touches on many diverse aspects of cellphysiology, including oxidant defense, cell wall remodeling,metabolism, and transport (Hahn et al. 2004). However,representatives from these various functional annotationsare few, making it unlikely that heat shock dramaticallyimpacts these processes. Rather, it may be that some ofthe proteins involved are thermolabile or perhaps rate lim-iting during heat shock, necessitating a transient increase ingene expression to support continued function under ther-mal duress. Transcriptional profiling revealed that a numberof duplicated isozymes are differentially regulated by stress(Gasch et al. 2000; Causton et al. 2001). For example, of thethree thioredoxins, TRX1, TRX2, and TRX3, only TRX2 issignificantly induced by stress. Likewise, one hexokinasegene, HXK1, is highly induced by stress, while the expres-sion of HXK2 remains largely unchanged. This theme isrecapitulated with the major HSPs: HSC82 (Hsp90) is con-stitutively expressed and abundant, while HSP82 is sig-nificantly expressed only during heat shock. Similarexpression patterns exist for Hsp70, Hsp110, and someHsp90 co-chaperones.

In contrast, genes encoding other HSPs such as Hsp104are abundant under normal growth conditions and substan-tially induced by heat shock. Finally, the significant overlapin expression programs observed among the different stres-sors that have been examined explains the phenomenon of“cross-protection,” whereby exposure to one stress enhancestolerance of a subsequent stress of a different nature; i.e.,heat shock induces tolerance to oxidants or osmotic shock.This is especially apparent for Msn2/4-dependent genes,most of which exhibit a common gene expression pattern

in response to diverse stressors, suggestive of a monolithic,rather than stress-specific response.

Cellular functions of the heat shock response and heatshock proteins

The preceding sections outlined advances in understandingcontrol and regulation of the HSR, with an emphasis ontranscription. While unstressed cells exhibit moderate re-sistance to a range of environmental stressors, mildlystressed cells significantly increase their ability to withstandfuture insult (Berry and Gasch 2008; Yamamoto et al.2008). One of the products of the HSR that confers cytopro-tection is the disaccharide trehalose. Work in the late 1990sidentified trehalose as a powerful stabilizer of proteins andmembranes in multiple biological systems, including yeast.The role of trehalose in heat shock survival has been ex-tensively reviewed elsewhere, and the reader is directedto these sources for additional information (Singer andLindquist 1998; Trott and Morano 2003; Crowe 2007).However it is the heat shock proteins that have receivedthe most attention to date, as they promote cell survivalunder both stress and nonstress conditions. The mechanisticdetails of protein chaperone functions at the molecular levelhave been the subject of intense scrutiny, and we havelearned much regarding their operations in vitro. In con-trast, a comprehensive understanding of the cellular rolesplayed by chaperones is only just emerging.

The yeast “chaperome”:With the advent of whole genomesequencing, the chaperone complement of organisms cannow be determined and defined as the chaperome(Morimoto 2008). This descriptor includes chaperones andother HSPs present under normal growth conditions as wellas those whose abundance increases, or are solely producedduring the HSR. Major HSPs were defined years ago on thebasis of their abundance during heat shock, when they areeasily detected by radioactive pulse–chase analysis becausetheir synthesis is increased while that of the remainder ofthe proteome is repressed (Subjeck et al. 1982). These HSPswere named according to their apparent molecular massesand gave rise to the by-now familiar collection of Hsp100,Hsp90, Hsp70, Hsp60, and the small HSPs ubiquitous ineukaryotic cells. Work over the last decade has uncoveredfew new chaperones in yeast; instead a panoply of chaper-one partner proteins, or “co-chaperones,” have been eluci-dated that contribute to HSP functions in numerous ways.Nowhere is this more apparent than for the Hsp70 andHsp90 chaperones, where work in yeast has led the wayin identifying important new co-chaperones that play rolesin protein folding, chaperone targeting, and substrateselection.

The Hsp70 chaperone system: The Hsp70 chaperone is the“workhorse” chaperone of eukaryotic cells [bacterial cellsrely to a greater extent on the Hsp60/chaperonin family(GroEL/ES)], that interacts with proteins at all stages in





















their lifetimes (Frydman 2001). Hsp70s protect nascent pol-ypeptides as they emerge from the ribosome, assist in target-ing and translocation, and play important roles in eitherrefolding damaged proteins or shepherding their ubiquityla-tion and degradation. Interactions with substrates occurthrough the C-terminal substrate binding domain, whoseaffinity for same is governed by allosteric movements inthe N-terminal nucleotide binding domain (Vogel et al.2006a,b). Hsp70•ATP loosely binds an extended, mostly hy-drophobic, polypeptide segment until nucleotide hydrolysis,after which substrate diffusion is restricted by conforma-tional shifts in the substrate binding domain (Jiang et al.2005). Protein folding is promoted by iterative cycles ofsubstrate binding and release until all hydrophobic regionsare suitably buried. Two classes of Hsp70 cofactors regulatethe speed of this cycle: diverse relatives of the bacterial DnaJco-chaperone termed J proteins, which enhance ATP hydro-lysis, and distinct classes of structurally unrelated proteinsthat act as nucleotide exchange factors.

As shown in Table 1, Hsp70s are found in the ER, mito-chondria, and cytoplasm. In the latter two compartments,a “specialized” Hsp70 operates alongside a “general” coun-terpart. In the mitochondrial matrix, Ssc1 is involved in pro-tein refolding after import, while Ssq1 is specificallyrequired for maturation of iron-sulfur cluster-containingproteins. Two specialized Hsp70s exist in the cytoplasmand both are localized to the ribosome; Ssb1/2 participatein folding of nascent chains, whereas the divergent Ssz1appears to have lost both nucleotide hydrolysis and sub-strate binding capabilities and instead promotes enhance-ment of Ssb1/2 function by the J protein Zuo1. Constitutive(Ssa1/2) and stress-inducible (Ssa3/4) Hsp70s carry outthe bulk of Hsp70 functions in the cytoplasm, while theKar2 protein plays the same role in the ER lumen. Twoeven more divergent Hsp70s exist in the ER (Lhs1)and cytoplasm (Sse1/2) and will be discussed below. Be-cause general Hsp70s such as the Ssa family exhibit pro-miscuous polypeptide binding and refolding activity, other

components must be responsible for conferring specificityand regulation.

On the basis of the highly conserved J domainsignature, �22 J proteins have been identified in S. cerevisiae(Kampinga and Craig 2010). These proteins are in generalhighly divergent outside the J domain and this diversity likelyplays an important role in determining Hsp70 involvement indistinct cellular processes. For example, Zuo1, involved intranslation, contains a ribosome-association domain that facil-itates interaction with Ssz1 and Ssb (Gautschi et al. 2001),and Swa2, required for endocytosis, contains a clathrin-binding domain (Gall et al. 2000). Some J proteins, suchas the major cytoplasmic J protein Ydj1, bind clients as wellas accelerate Hsp70 ATP hydrolysis, while others bind spe-cific clients only or not at all. J proteins also localize todifferent cellular locations, including the mitochondrion,ER lumen, and ER membrane, where they presumably in-crease the local concentration of Hsp70 activity to promotespecific cellular processes. A good example of this is theSec63 protein, which operates with the Sec61 transloconchannel in protein import and insertion into the ER. Sec63contains a J domain localized within the ER lumen thatpromotes recruitment and function of Kar2 to catalyze pro-tein translocation (Feldheim et al. 1992). A similar case canbe made for Swa2 (Aux1), which recruits Ssa to clathrin-coated vesicles where it participates in disassembly anduncoating (Gall et al. 2000). The J domain can thereforebe considered an Hsp70 recruitment module that serves tointegrate this powerful protein remodeler into cellularactivities.

Four classes of nucleotide exchange factor (NEF) havebeen uncovered in yeast with orthologous human counter-parts, and unlike the J proteins, they bear no sequence orstructural homology with each other. One class is found onlyin the mitochondrial matrix, encoded by MGE1 and resem-bling the GrpE NEF from Escherichia coli (Laloraya et al.1994; Voos et al. 1994). The yeast homolog of the Bagfamily of human NEFs, termed Snl1, is tethered to the ER

Table 1 Hsp70 chaperones in Saccharomyces cerevisiae

Gene name Localization Function Reference

Ssa1 Cytosol General folding Werner-Washburne et al. (1987)Ssa2 Cytosol General folding Werner-Washburne et al. (1987)Ssa3 Cytosol General folding Werner-Washburne et al. (1987)Ssa4 Cytosol General folding Werner-Washburne et al. (1987)Ssb1 Ribosome Nascent chain folding Nelson et al. (1992)Ssb2 Ribosome Nascent chain folding Nelson et al. (1992)Ssz1 Ribosome Nascent chain folding Gautschi et al. (2002); Hundley et al. (2002)Sse1 Cytosol Substrate binding; NEF Dragovic et al. (2006); Raviol et al. (2006); Shaner et al. (2006)Sse2 Cytosol Substrate binding; NEF Dragovic et al. (2006); Raviol et al. (2006); Shaner et al. (2006)Kar2 ER General folding Vogel et al. (1990)Lhs1 ER Substrate binding; NEF Baxter et al. (1996); Craven et al. (1996);

Hamilton and Flynn (1996); Steel et al. (2004)Ssc1 Mitochondrion Postimport folding Craig et al. (1989)Ssq1 Mitochondrion Assembly Fe/S proteins Dutkiewicz et al. (2003)Ecm10 Mitochondrion Postimport folding Baumann et al. (2000)






























membrane with its Bag domain facing the cytoplasm (Hoet al. 1998; Sondermann et al. 2002). Two yeast proteinsare related to the human HSPBP1 NEF: Sls1 localized to theER lumen (Kabani et al. 2000) and Fes1, which resides inthe cytoplasm (Kabani et al. 2002). The most unusual groupconsists of the Lhs1 protein in the ER lumen (Craven et al.1996; Saris et al. 1997) and the Sse1/2 proteins in thecytoplasm (Mukai et al. 1993), both of which share thegeneral domain architecture of the typical Hsp70s withinsertions and C-terminal extensions that increase their mo-lecular mass leading to their categorization as members ofthe “Hsp110” family (based on the size of the human ortho-logs) (Easton et al. 2000; Shaner and Morano 2007). On thebasis of in vitro studies, these proteins are incapable of fold-ing substrates on their own and instead have evolved a novelHsp70 binding interface, which they use to form highly sta-ble Hsp70•Hsp110 heterodimers to enhance nucleotide ex-change (Shaner et al. 2005; Yam et al. 2005; Dragovic et al.2006; Raviol et al. 2006; Polier et al. 2008; Schuermannet al. 2008). However, the peptide binding domains of theHsp110 family, while altered with respect to Hsp70, retainthe ability to bind and protect unfolded polypeptides asexemplified by the yeast Ssa1 protein (Brodsky et al.1999). This raises the possibility that this group may act ina manner similar to the client-binding J proteins to enhancesubstrate delivery and transfer to Hsp70 for subsequent fold-ing. Unlike the J proteins that have been implicated in spe-cific processes via genetic and biochemical interactions, nosuch targeted or exclusive activities have been described forthe NEFs. However the fact that NEF activity has arisenmultiple times in evolution strongly hints that the co-chap-erones are likely to play nonredundant roles.

The Hsp90 chaperone system: In contrast to the Hsp70chaperone system, which interacts with nearly any partiallyunfolded protein it encounters, the Hsp90 chaperone ismuch more selective and interacts with a small but growinglist of protein “clients.” These clients rely on Hsp90 for thefinal steps of maturation after initial interactions withHsp70, effectively linking these two chaperone machinesin an “assembly line” of protein maturation. Hsp90 is highly

abundant, accounting for 1–2% of total protein during stressconditions (Borkovich et al. 1989). As with Hsp70, Hsp90 isan ATP binding protein whose chaperone cycle is governedby nucleotide cycling (see Taipale et al. 2010). Also analo-gous to Hsp70, a sizable number of co-chaperones regulatesteps in the nucleotide cycle in yeast and other eukaryotes.Although a comprehensive treatment is beyond the scope ofthis chapter, these co-chaperones can be grouped into threeclasses: those that bind the ATPase domain of Hsp90, theAha1 protein that binds the middle domain, and a handfulof proteins containing tetratricopeptide repeats (TPRs) thatbind the highly conserved MEEVD sequence at the extremeC terminus of Hsp90 (see Table 2). A canonical client mat-uration cycle has been elucidated in vitro for animal steroidhormone receptors with the “minimal” complement ofHsp70/Hsp40, the linker protein HOP (homologous to theTPR-containing proteins Sti1 and Cns1), Hsp90, and theprotein p23 (yeast Sba1) that stabilizes the ATP-bound state(Dittmar et al. 1997). However, variations on this theme forother clients suggest that the exceptions may outnumber therule. For example, many if not all protein kinases interactwith Hsp90 at some point in their maturation but do so inconcert with the protein Cdc37, which can partner withHsp90 or promote kinase maturation independently (Leeet al. 2002; Mandal et al. 2007). In contrast, most otherHsp90 clients do not require Cdc37 for their activity. Theimmunophilin homologs Cpr6 and Cpr7 likewise play littleto no role in kinase maturation yet are required for otherHsp90 activities such as steroid receptor activity and Hsf1repression (Duina et al. 1996, 1998). Co-chaperones canalso link Hsp90 to specific pathways, as the p23-like proteinSgt1 associates with both Hsp90 and the Skp1–Cul1–F-box-protein (SCF) ubiquitin ligase complex through the multi-functional kinetochore protein Skp1 (Kitagawa et al. 1999).Hsp90 activity is also modulated by phosphorylation inyeast, mediated by the TPR-containing protein phosphatasePpt1 and the tyrosine kinase Swe1 (Wandinger et al. 2006;Mollapour et al. 2010).

Although only a small number of yeast Hsp90 clientshave been experimentally verified, genomic and proteomicapproaches are being used to identify novel clients with the

Table 2 Hsp90 co-chaperones in Saccharomyces cerevisiae

Gene name Function Reference

STI1 TPR; Hsp70/Hsp90 bridging Chang et al. (1997)CNS1 TPR; unknown Dolinski et al. (1998)PPT1 TPR; Hsp90 regulation Wandinger et al. (2006)TOM70 TPR; mitochondrial import Young et al. (2003)CPR6 TPR; Hsp90 regulation Duina et al. (1996)CPR7 TPR; Hsp90 regulation Duina et al. (1996)AHA1 Hsp90 ATPase regulation Panaretou et al. (2002)SGT1 Targeting Hsp90 to kinetochore, SCF complex Kitagawa et al. (1999)SBA1 Hsp90 ATPase regulation Bohen 1998; Fang et al. (1998)CDC37 Kinase targeting chaperone Dey et al. (1996)TAH1 Targeting Hsp90 to Rvb1/2 complex Zhao et al. (2005a)





















goal of trying to establish the complete substrate comple-ment in a single organism (Zhao et al. 2005b). Two majorcriteria must be satisfied to warrant inclusion on the list:clients must associate with Hsp90 at some point in theirlifetime and pharmacological (with potent and specificHsp90 ATPase inhibitors) or genetic depletion must affectthe protein’s stability and or cellular function. The Picardlaboratory in Geneva, Switzerland maintains a list of clientsthat is continuously updated and contains a number of en-dogenous yeast proteins (http://www.picard.ch/downloads).

Oligomeric HSPs: Hsp104 and the small HSPs: In contrastto the previously described chaperones, the yeast proteinHsp104 and a series of small HSPs function in vivo andin vitro as oligomeric complexes. Hsp104 is a member ofthe AAA+ ATPase family, which includes the well-describedbacterial Clp chaperones/proteases as well as nonchaperoneproteins Sec18 (vesicle fusion) and Cdc48 (ERAD), whichform hexameric rings with a large (�15 Å) central channel(Doyle and Wickner 2009). Unlike most other chaperones,Hsp104 is capable of extracting misfolded proteins fromaggregates, followed by translocation through the centralchannel. Hsp104 cannot refold proteins alone, however,and relies on Hsp70 to fully rescue substrates and returnthem to the appropriate native conformation. In addition,Hsp70 likely participates in early steps of recognition andperhaps priming of polypeptide extraction, making theHsp104/Hsp70 partnership nearly obligatory (Parsell et al.1994). Hsp104 is also one of the few yeast protein chaper-ones absolutely required for thermotolerance: hsp104Δ cellsare exquisitely sensitive to lethal heat shock (Sanchez andLindquist 1990). This protection comes at a price, as Hsp104contains two independent but linked ATPase domains thatbind and hydrolyze up to 12 ATP molecules per cycle, withpotentially hundreds of cycles required per extracted protein(Doyle and Wickner 2009). Interestingly, humans lackHsp104 or analogous disaggregase activity, although yeastHsp104 expressed in human cells confers thermoprotec-tion (Chernoff et al. 1995; Mosser et al. 2004). Hsp104is also a primary modulator of yeast prion/amyloid stabil-ity and inheritance, where it is involved in processingfibers, which ultimately generates additional prion “seeds”that enhance distribution and substrate conversion (Chernoffet al. 1995).

Small HSPs (sHSPs) represent a diverse family of proteinswith passive chaperone activity that are classed together dueto sequence similarity with the eye lens protein a-crystallin.All known sHSPs exist either transiently or stably in highmolecular weight oligomeric structures, where they inter-act with unfolded substrates usually at a 1:1 monomer-to-substrate ratio (Wotton et al. 1996). Instead of preventingthe aggregation of damaged proteins, sHSPs appear to “co-aggregate” with their substrates in a mixed oligomeric ag-glomeration that can be resolubilized with the help of addi-tional chaperones (Haslbeck et al. 2005). In yeast, twomajor sHSPs have been characterized, Hsp26 and Hsp42.

Hsp42 is expressed in unstressed cells and forms a large,stable, barrel-like oligomer, whereas Hsp26 undergoes a dy-namic transition as part of its chaperone activity (Haslbecket al. 1999; Stromer et al. 2004). Under normal tempera-tures, Hsp26 exists as a 24-mer but rapidly dissociates uponheat shock via a novel thermosensing domain into dimersthat are capable of interacting with unfolded substrates(Stromer et al. 2004). Hsp26:substrate oligomers then reas-semble into a novel heterooligomer. Refolding of sHSP-asso-ciated substrates requires the action of the ATP-dependentHsp70 and Hsp104 chaperones, which extract polypeptidesfrom their protected state in the aggregate (Haslbeck et al.2005). Both Hsp26 and Hsp42 are required to maintain theyeast proteome in a soluble state during heat shock, al-though Hsp26 appears to play a limited role under normalgrowth conditions (Haslbeck et al. 2004). In spite of thedramatic transfer of a significant fraction of cellular proteinsfrom a soluble and presumably functional state to an insol-uble, aggregated state in hsp26Δ and hsp42Δ mutants, noobvious growth phenotypes have been reported. However,examination of these mutants via scanning electron micros-copy reveals profound morphological cellular surface alter-ations reminiscent of aged or dehydrated cells (Haslbecket al. 2004). This “wrinkled” phenotype is also shared bycells lacking another poorly understood low-molecularweight HSP unrelated to the sHSP family, Hsp12. Hsp12 isa highly abundant protein that exists in a mostly unfoldedstate in its soluble form, although a subset of the total Hsp12pool associates with cellular membranes, where it acquiresa helical structure and increases membrane stability (Welkeret al. 2010). Not surprisingly, hsp12Δ cells are hypersensitiveto severe heat shock and osmotic stress, demonstrating thatthe chaperone plays an important role in membrane protec-tion (Welker et al. 2010).

Chaperone networks: The yeast genome contains at least60 $known chaperones: 7 sHSPs, 14 belonging to the CCT/TRiC and prefoldin complexes, 2 Hsp90s, 14 Hsp70s, 1Hsp60, 3 AAA+ ATPases (Hsp104), and 22 Hsp40s (Gonget al. 2009). How is the action of all these protein remod-elers orchestrated at the cellular level? A combination oftranscriptome profiling, proteomics, and large-scale pheno-typic analysis suggests that the majority of protein chaper-one function in the yeast cell can be divided into two classes:those that participate in protein translation and nascentchain folding and those required for repair and recoveryafter severe stress (Albanese et al. 2006). The former group,termed chaperones linked to protein synthesis (CLIPS),physically associate with ribosomes, are transcriptionallydownregulated during stress along with RPs, and when dis-rupted confer profound sensitivity to translation inhibitorssuch as hygromycin and cycloheximide but not to heatshock. In contrast, the HSPs are induced by heat shockand are required for survival after severe thermal stress.These categories are obviously not completely mutually ex-clusive, but the degree to which phenotypes are shared


http://www.picard.ch/downloads
































within the two classes is remarkable. The overlap betweenthe CLIPS and HSPs is shown schematically in Figure 3.

The intersection between the HSR and otherstress responses

HSR and oxidative stress response: The HSR, or morebroadly defined the ESR, includes the reprogramming ofa significant percentage of the total transcriptome. Thissuggests that heat shock impacts more than just proteinstability. Indeed, the heat shock-induced ESR includes genesinvolved in metabolism, oxidant defense, and growthcontrol. Tolerance of severe heat shock is in fact tightlylinked to aerobic metabolism and oxidative stress. Yeastcells cultured anaerobically are 102- to 103-fold more resis-tant to heat shock than those grown in the presence ofoxygen (Davidson et al. 1996). Cells lacking antioxidantenzymes such as catalase and superoxide dismutase (SOD)are conversely hypersensitive to heat shock (Davidson et al.1996). Levels of reduced glutathione decline during aerobic,but not anaerobic, heat shock (Sugiyama et al. 2000b).These results suggest that the primary stress of heat shockinduces a subsequent oxidative stress as a function of oxy-gen availability. In an aerobically growing cell, the main fluxof oxygen is through the electron transport system localizedto the mitochondrial network. A test of the hypothesis thatheat induces oxidative stress by disruption of electron flowthrough the respiratory chain through deletion of the COQ7gene (encoding coenzyme Q) revealed increased heat sen-sitivity and nuclear mutation frequency (Davidson andSchiestl 2001). These effects are blocked by deletion ofthe NADH dehydrogenases NDE1 and NDE2, required forelectron transfer from NADH to the respiratory chain(Davidson and Schiestl 2001). Heat-induced oxidative stressis countered by induction of antioxidant genes, including

the glutathione biosynthesis genes GSH1 and GSH2, ina Yap1-dependent manner only under aerobic conditions(Sugiyama et al. 2000a). This conditional regulation allbut guarantees that it is not the thermal stress but insteadthe resulting oxidative stress that is the proximal inducer ofantioxidant gene transcription. The mitochondrial Mg2+-SOD encoded by SOD2 (also an ESR gene) is also requiredfor tolerance to heat-induced oxidative stress (Sugiyamaet al. 2000b). A recent study demonstrated that both Hsf1and Yap1 are activated by the natural product celastrol, as isthe case with the heavy metal cadmium, suggesting thatadequate cellular defense against some noxious agentsrequires coordination of these two pathways (Trott et al.2008). Although the mechanism behind this synchrony isunclear, it is interesting to note that another major mediatorof the oxidative stress response (OSR) is the Skn7 transcrip-tion factor whose DNA binding domain is highly similar tothat of Hsf1 (Morgan et al. 1997; Lee et al. 1999a). Therelevance of the interaction between Skn7 and Hsf1 as itpertains to transcriptional induction in response to heatand oxidants is discussed later (Transcription control of theOSR). Superoxide anion selectively activates Hsf1 expres-sion of the copper metallothionein CUP1 and was shownto induce Hsf1 binding to DNA in cell-free extracts, similarto mammalian HSF1, which responds to treatment with hy-drogen peroxide (Liu and Thiele 1996; Lee et al. 2000; Ahnand Thiele 2003). These findings demonstrate that HSFs cansense oxidative stressors. However, mammalian HSF1 con-tains two cysteine residues located within the DNA bindingdomain that mediate activation through the reversible for-mation of a disulfide bond, whereas yeast Hsf1 lacks anycysteine residues (Ahn and Thiele 2003). How then is yeastHsf1 regulated by oxidants? It is likely that one or more cel-lular components acts as a sensor for thiol-reactive molecules,supported by the observation that glutathione treatment blockssuperoxide-activated DNA binding by Hsf1 in cellular extracts,but not of purified Hsf1 (Lee et al. 2000). Identification ofthese factors will be important for resolving the molecularmechanisms behind this disparity and understanding the evo-lution of HSF as an ancillary component of the OSR.

HSR and cell wall integrity: The cell wall integrity (CWI)pathway responds to perturbations of the cell wall viaactivation of a MAP kinase pathway by plasma membrane-localized sensor proteins (reviewed thoroughly in Levin2005). This pathway is also induced by transient heat shockor growth at 37�, suggesting that such temperatures desta-bilize the membrane or cell wall. Several Hsf1 and chaper-one mutants retain viability at 30� but are temperaturesensitive for growth at 37� (Zarzov et al. 1997; Moranoet al. 1999; Shaner et al. 2008). Surprisingly, these pheno-types are apparently linked as both can be reversed by sup-plementation of the growth medium with 1 M sorbitol, anosmostabilizer that also suppresses mutants in the CWIpathway. This effect can be traced to defects in the terminalMAP kinase of the CWI pathway, Slt2 (Mpk1), a client of

Figure 3 CLIPs and HSPS. Venn diagram depicting the intersection be-tween chaperone networks, based on the work of Frydman and cow-orkers, is shown (Albanese et al. 2006). See text for details.

























Hsp90 that exhibits reduced activity in strains pharmacolog-ically or genetically manipulated to be deficient in Hsp90activity (Millson et al. 2005; Truman et al. 2007). Overex-pression of Slt2, Hsp90, or the Slt2-dependent transcriptionfactor Rlm1 also suppresses the temperature-sensitive phe-notype of Hsf1 mutants, confirming that activation of theCWI pathway is necessary and sufficient to confer 37�growth (Truman et al. 2007). These findings challenge theconventional wisdom that strains defective in the HSR andprotein chaperone function are thermosensitive due to pro-teome-wide folding defects. Instead, most if not all of the37� growth phenotypes (but importantly not those due tosevere heat shock at temperatures above 45�) can be attrib-uted to dysfunction of the CWI pathway due to the depen-dence of Slt2 activity on the Hsp90 system.

Yeast as a model system for understanding the HSR

Exploiting yeast to understand the human HSR: S. cerevi-siae has served as an ideal model system to dissect the in-tricacies of the eukaryotic HSR, which while thematicallysimilar to the bacterial response to heat shock differs interms of players and regulation. The ease of genetic manip-ulation and more recently advances in genomic transcrip-tional profiling and proteomic analyses has allowed a paceof discovery and investigation not possible in human cells.However, certain features of the human HSR are not repre-sented in the yeast model. For example, as described earlier,yeast Hsf1 is constitutively trimerized and bound to manyHS promoters in contrast to the situation in mammalian cellswhere HSF1 is maintained as an inactive monomer in thecytoplasm. In addition, while mammals express multipleHSF isoforms with distinct functions, fungi possess onlya single Hsf1. Post-translational regulation of Hsf1 likelydiffers substantially between yeast and human cells. Lastly,higher eukaryotes do not possess a broad ESR mediated bynon-Hsf1 factors. These disparities have been exploited toutilize yeast as a test system to understand intricacies ofhuman HSF1 (hHSF1) function. Human HSF2, but nothHSF1, functionally complements a yeast hsf1Δ null strain,demonstrating that the hHSF2 isoform, which regulates de-velopmentally controlled genes, retains its ancestral abilityto control HSP genes (Liu et al. 1997). hHSF1 can be madefunctionally competent in yeast through disruption ofa coiled-coil intramolecular interaction domain or substitu-tions within the DBD, suggesting that human cells normallymodulate these interactions to allow stress-induced activa-tion (Liu and Thiele 1999; Takemori et al. 2009). Analysis ofhHSF1 in the yeast system has also revealed that a loopregion within the highly conserved DNA binding domaininfluences promoter recognition and trimerization (Ahnet al. 2001). In a recent exciting development, the comple-mentation system has been used as a drug discovery plat-form to identify a new class of HSR-activating compounds(Neef et al. 2010). These advances are of significant rele-

vance to human health as modulation of the HSR showspromise for the treatment of diseases linked to protein fold-ing such as neurodegenerative disorders (e.g., Alzheimer’sdisease, Parkinson’s disease, Lou Gehrig’s disease) (Westerheideand Morimoto 2005). In addition, the collapse of proteinhomeostasis as organisms age is increasingly being recog-nized as a contributing factor to the pathologies of aging(Morimoto 2008). Restoration of protein biogenesis and re-pair through pharmacologic activation of the HSR is an in-triguing possibility that will rely on mechanistic informationobtained from yeast and other model eukaryotic systems.

The HSR in other yeasts: Analysis of the HSR in pathogenicfungi has followed closely on the heels of its elucidation inS. cerevisiae. Significant effort in understanding the HSR in thepathogenic, dimorphic yeast Candida albicans has revealedthat the organism lacks a general stress response akin to thebaker’s yeast ESR (Enjalbert et al. 2003). This is consistentwith work showing that the closest homologs of Msn2/4 inC. albicans play no role in stress resistance, nor do theycontribute to the stress transcriptome (Nicholls et al.2004). The question of whether an obligate animal patho-gen requires a robust HSR is a relevant one, as C. albicansenjoys a controlled environment of 37�, with only occa-sional, modest increases in temperature (the febrile state).In fact, C. albicans exhibits an abridged HSR centered on themajor HSPs and a handful of OSR genes, most of which arevery modestly induced (two- to sixfold) relative to theirbaker’s yeast counterparts (Enjalbert et al. 2003). Notablyabsent are the major changes in carbohydrate metabolismthat promote sugar storage. C. albicans Hsf1 displays most ofthe characteristics of the S. cerevisiae protein and appears toplay a major role in basal expression of key HSPs, includingHsp70 and Hsp90. Hsf1 is required for virulence, at least inpart through its ability to induce HSP expression duringstress, but potentially due to the requirement for basal levelsof Hsp70/Hsp90 production (Nicholls et al. 2009, 2010).This distinction is difficult since Hsp90 (and by extension,Hsp70 due to extensive collaboration between the two chap-erone machines) is likely required for signaling during in-fection. Moreover, Hsp90 has been identified as a keymediator of fungal drug resistance in C. albicans and Asper-gillus fumigatus (see Cowen 2009 for a summary). This hasprompted work to exploit the potential of therapeutic syn-ergy between antifungals such as the azoles and the newerechinocandins and Hsp90 inhibitors such as geldanamycinand its derivatives (Cowen et al. 2009; Singh et al. 2009).

The Oxidative Stress Response

While elevated temperature represents the primary insultduring heat shock (as described above), one of the majorsecondary consequences involves production of reactiveoxygen species (ROS). All organisms are exposed to ROSduring the course of normal aerobic metabolism or followingexposure to radical-generating compounds (Halliwell 2006).














Molecular oxygen is relatively unreactive and harmless in itsground state, but can undergo partial reduction to forma number of ROS, including the superoxide anion and hy-drogen peroxide (H2O2), which can further react to producethe highly reactive hydroxyl radical (Figure 4). ROS aretoxic agents that can damage a wide variety of cellular com-ponents resulting in lipid peroxidation, protein oxidation,and genetic damage through the modification of DNA. Anoxidative stress is said to occur when the antioxidant andcellular survival mechanisms are unable to cope with theROS or the damage caused by them (Figure 5). Variousdisease processes, including cancer, cardiovascular disease,arthritis, and aging have been shown to involve oxidativedamage. Oxidative damage is also of particular concern toindustry, since the oxidation of fats and oils is the processunderlying food rancidity, and yeast cells used in the bakingand brewing industries are exposed to oxidative stressesduring freezing and drying. S. cerevisiae responds to an ox-idative stress using a number of cellular responses that en-sure the survival of the cell following exposure to oxidants.These include defense systems that detoxify ROS, reducetheir rate of production, and repair the damage caused bythem. Many responses are ROS specific, but there are alsogeneral stress responses that are typically invoked in re-sponse to diverse stress conditions.

Sources of ROS generation in yeast cells

ROS generation naturally arises from environmental insultsand from side reactions of normal aerobic metabolism.Mitochondrial respiration is thought to provide the mainsource of ROS in eukaryotic cells via the process of oxidativephosphorylation (Murphy 2009). To generate ATP, electronsare transported along protein complexes that constitute theelectron transport chain to the ultimate acceptor, molecularoxygen, with the formation of water. Leakage of these elec-trons from the respiratory chain can result in the reduction ofoxygen, generating ROS in yeast cells (Figure 5). Similarly,

the use of oxygen as a terminal electron acceptor duringoxidative protein folding means that the ER is also a signifi-cant source of ROS (Tu and Weissman 2004). Other meta-bolic processes that can potentially generate endogenousROS in yeast, depending on the growth conditions, includeperoxisomal fatty acid degradation in the b-oxidation path-way (Hiltunen et al. 2003) and oxidative deamination ofamino acids by D-amino acid oxidases during growth on D-amino acids as carbon sources (Pollegioni et al. 2007). Yeastcells can also become exposed to ROS produced by neutro-phils and macrophages during immunological defense mech-anisms and following exposure to numerous exogenousagents including xenobiotics, carcinogens, and UV and ioniz-ing radiation (Halliwell 2006).

Commonly used model ROS compounds

Many oxidative stress studies have made use of singlecompounds such as hydrogen peroxide (H2O2) as a modeloxidant. However, given that cells can respond to ROS viaoxidant-specific responses, it has been argued that no singleoxidant can truly be said to be representative of “oxidativestress” (Temple et al. 2005). Genome-wide screens of theyeast deletion collection have examined different ROS andfurther shown that there are both core functions that arerequired for a broad range of oxidative-stress conditions aswell as ROS-specific functions that are only required duringparticular oxidant conditions (Thorpe et al. 2004). A briefdiscussion of the more frequently used model ROS com-pounds is provided here.

Hydroperoxides: Due to its ease of use including watersolubility and relative stability, H2O2 is most widely used as

Figure 4 Generation of ROS. The superoxide anion (O22) can be formed

via electron leakage to oxygen from electron transport chains. Hydrogenperoxide (H2O2) is generated by the breakdown of superoxide catalyzedby superoxide dismutases (SODs). Hydrogen peroxide can be reduced by iron(Fe2+) in the Fenton reaction to produce the highly reactive hydroxyl radical.In the Haber-Weiss reaction, superoxide can donate an electron to iron (Fe3+),generating the hydroxyl radical and Fe2+, which can further reduce hydrogenperoxide. Various antioxidant enzymes, including catalases and peroxidases,detoxify hydrogen peroxide to prevent such ROS generation.

Figure 5 Oxidative stress. All organisms can be exposed to ROS duringthe course of aerobic metabolism or following exposure to ionizing radi-ation and radical-generating compounds. Antioxidant defense systemsprotect against ROS by detoxifying ROS as they are generated and bymaintaining the intracellular redox environment in a reduced state. Anoxidative stress occurs when the antioxidant and cellular survival mecha-nisms are unable to cope with the ROS or the damage caused by them.Oxidative stress can damage a wide variety of cellular components result-ing in lipid peroxidation, protein oxidation, and genetic damage throughthe modification of DNA.


a model oxidative stress condition. H2O2 is a ubiquitousmolecule formed as a byproduct of aerobic respiration andfollowing exposure to diverse biological and environmentalfactors. It can damage cells by promoting oxidative stressbut also plays important roles as a signaling molecule inthe regulation of many biological processes (Veal et al.2007). H2O2 must be removed from cells to avoid Fentonand Haber-Weiss reactions leading to the formation ofhighly reactive hydroxyl radicals (OH) (Figure 4). Organichydroperoxides are often used to invoke lipid oxidation. Forexample, cumene hydroperoxide (C9H12O2) is an aromaticlipid soluble hydroperoxide that is widely used as an intra-cellular source of ROS (Thorpe et al. 2004). It can generatehighly reactive free radicals such as the alkoxy radical,resulting in high mutagenicity and toxicity (Simic et al.1989). tert-butyl hydroperoxide ((CH3)3COOH) is less hy-drophilic than hydrogen peroxide and is frequently used asa model alkyl hydroperoxide. Linoleic acid hydroperoxide(LoaOOH) has been used as a model lipid hydroperoxidein yeast and is toxic to yeast cells at very low concentrationscompared to H2O2 and other organic peroxides (Evans et al.1997). The importance of examining diverse hydroperoxidesis emphasized by recent findings that indicate that hydrogenperoxide and tert-butyl hydroperoxide induce different cel-lular signaling responses (Iwai et al. 2010).

The superoxide anion: The superoxide anion (O22) is gen-

erated by one electron reduction of oxygen (Figure 4). It isthe major ROS product resulting from electron leakage fromthe mitochondrial electron transport chain (Halliwell 2006).The superoxide anion is not highly reactive itself but can actas a precursor for other ROS via dismutation to hydrogenperoxide and can generate the highly reactive hydroxyl rad-ical via metal-catalyzed reactions. The superoxide anion canreadily be generated in yeast cells using redox-cycling drugssuch as menadione and paraquat, which transfer electronsto molecular oxygen.

Thiol-reactive compounds: Given the importance of redoxhomeostasis during oxidative stress conditions, thiol-reac-tive compounds are frequently used to induce oxidativestress. These include compounds that indirectly cause anoxidative stress by binding to and depleting thiol groups, aswell as compounds which directly oxidize thiol groups. Forexample, 1-chloro-2,4-dintrobenzene (CDNB) is a substratefor glutathione transferases (Sheehan et al. 2001). It doesnot oxidize thiols in yeast but causes oxidative stress bydepleting cellular glutathione (GSH), presumably leadingto an accumulation of endogenous ROS (Wheeler et al.2002). Diamide is a membrane-permeable, thiol-specificoxidant, which promotes the formation of disulfides(Kosower and Kosower 1995). It has frequently been usedto induce oxidative stress in yeast where it causes a rapidoxidation of glutathione that shifts the redox state of theglutathione redox couple to a more oxidized form (Muller1996).

Heavy metal stress: The availability of free redox activemetals such as iron and copper can have a profoundinfluence on the generation of cellular ROS (Liochev andFridovich 1999). For example, reduced Fe2+ can generatethe highly reactive hydroxyl radical via the Fenton reaction(Figure 4). Oxidized Fe3+ can be reduced by the superoxideanion further exacerbating the production of hydroxyl rad-icals in the Haber-Weiss reaction. Hence it is particularlyimportant that free metal levels are tightly controlled, butthis is confounded by oxidant attack of [4Fe-4S] clusters inproteins that can themselves provide a source of free iron(De Freitas et al. 2003). Cadmium is a highly toxic metal anda well-established human carcinogen, which is often inter-preted as causing an oxidative stress (Brennan and Schiestl1996). It is capable of entering cells via the same transportsystems used by the essential heavy metals. Once inside thecell, the main mechanism for toxicity is through the deple-tion of GSH and binding to sulfhydryl groups (Wysocki andTamas 2010). It can also displace iron and copper fromvarious cytoplasmic and membrane proteins, increasingthe levels of unbound free copper and iron ions, contributingto oxidative stress via Fenton reactions.

Transcriptional control of the OSR

Mounting the defensive response to elevated levels of ROS isa crucial step in preventing cell death from loss ofphysiologically appropriate redox balance. A key feature inthis response is the transcriptional reprogramming of geneexpression to provide the requisite changes in proteins toreturn the redox status of the cell back to an acceptablerange. Predictably then, transcriptional regulators that leadto induction of antioxidant proteins have been identifiedand the focus of much study.

Yap1: A primary determinant in the antioxidant response isthe transcription factor Yap1 (Harshman et al. 1988). Thisbasic region-leucine zipper-containing positive transcrip-tional regulator was the second member of the bZip tran-scription factors discovered in S. cerevisiae, after its relativeGcn4 (Harshman et al. 1988; Jones et al. 1988). Early workestablished that Yap1 was likely to be a positive regulator ofgene expression (Harshman et al. 1988; Jones et al. 1988)and was capable of conferring a multiple or pleiotropic drugresistance phenotype when overproduced (Leppert et al.1990; Hussain and Lenard 1991). Several groups found thatYap1 was critical for tolerance to oxidants such as H2O2 anddiamide, as well as heavy metals like cadmium (Schnell andEntian 1991; Kuge and Jones 1994; Wu and Moye-Rowley1994). These data provided the first association of Yap1 andits central role as a determinant of oxidative stress tolerance.

Nuclear localization of Yap1 responds to oxidative stress:Genetic evidence provided a clear linkage between thepresence of Yap1 and normal resistance to oxidants. Elegantexperiments using the then recently developed green fluo-rescent protein (GFP) determined that in the absence of








oxidants, Yap1 was found primarily in the cytoplasm (Kugeet al. 1997). Challenge of the cells with diamide led to therapid accumulation of Yap1 in the nucleus, with concomi-tant induction of target gene expression. The carboxy termi-nus of Yap1 was found to be both necessary and sufficientfor diamide-induced Yap1 nuclear localization. Three of thesix cysteines contained in Yap1 were present in this C-terminaldomain, which was designated the cysteine-rich domain(CRD) in recognition of the relative enrichment of this redoxactive amino acid.

While these data provided a consistent and simpleexplanation for control of Yap1 activity by diamide, regula-tion of this factor during H2O2 stress was more complex.Loss of the carboxy terminus of Yap1 led to a hypersensitivephenotype when these strains were challenged with H2O2

(Wemmie et al. 1997). Additionally, deletion mutant deriv-atives lacking the second CRD located in the amino terminusof the factor were diamide hyperresistant but H2O2 hyper-sensitive. Together, these data indicate that different seg-ments of Yap1 are required for this transcription factor tocarry out its normal function in the presence of these differ-ent oxidants.

Work on the Schizosaccharomyces pombe Yap1 homologcalled pap1+ provided the first demonstration of the pres-ence of a trans-regulator of these bZip transcription factors(Toda et al. 1991, 1992). Crm1 was first found as a factorrequired for normal chromosome structure (Adachi andYanagida 1989). This same hypomorphic allele of CRM1exhibited a staurosporine hyperresistant phenotype, whichwas ultimately demonstrated to be due to localization ofpap1+ to the nucleus (Toone et al. 1998). Experiments inS. cerevisiae showed that Crm1 in this yeast was also re-quired for nuclear export of Yap1 under nonstressed condi-tions (Kuge et al. 1998; Yan et al. 1998). Biochemicalexperiments localized the region of Yap1 required to asso-ciate with Crm1 to the CRD segment of the transcriptionfactor. This association required the presence of reducedcysteine residues in the CRD. Oxidation or replacement ofcysteine residues with other amino acids blocked binding ofYap1 and Crm1, correlating with constitutive nuclear local-ization of Yap1. This work led to the key conclusion thatretention of Yap1 in nucleus in response to oxidant exposurewas the result of control of nuclear export of Yap1, ratherthan the direct regulation of Yap1 import. Later work map-ping the bipartite nuclear localization signal of Yap1 to itsextreme amino terminus confirmed this view of the centralimportance of nuclear export in control of Yap1 activity (Iso-yama et al. 2001).

Most of the studies analyzing regulated nuclear traffick-ing of Yap1 employed diamide as the oxidant to trigger theresponse of this protein. While mutagenesis experimentsdemonstrated that mutant derivatives of Yap1 exhibited dra-matically different phenotypes in the presence of diamide vs.H2O2, the basis for this oxidant-specific response of Yap1was unknown. This murky view began to change with theconsideration of the thioredoxin-encoding gene TRX2 as

a key determinant in the Yap1-dependent induction ofH2O2 resistance.

TRX2 had already been found to be required for H2O2

resistance and to be responsive to Yap1 (Kuge and Jones1994). Analysis of a range of mutant forms of Yap1 indi-cated that alterations in the CRD caused constitutive activa-tion of an artificial Yap1-responsive reporter gene butprevented the ability of these same mutants to supportH2O2-induced TRX2 transcription (Coleman et al. 1999).These observations indicated that CRD mutants were ableto elevate the expression of genes involved in diamide re-sistance but other genes (such as TRX2) placed additionalrequirements on Yap1 for transcriptional activation. Furtherexperiments in this study provided direct evidence that theamino-terminal cluster of cysteine residues were also re-quired for response to H2O2. The two cysteine-rich domainswere then designated the n-CRD and c-CRD to denote theirrelative amino- or carboxy-terminal location.

While the presence of both CRD regions was necessaryfor normal H2O2 regulation of TRX2 transcription, the mo-lecular mechanism underlying this bipartite requirementremained unknown. Direct analysis of oxidative folding ofYap1 provided crucial insight into the roles of the n- andc-CRD regions (Delaunay et al. 2000). Use of nonreducingSDS–PAGE demonstrated that a disulfide bond could formbetween the two CRDs consisting of cysteine 303 (located inthe n-CRD) and cysteine 598 (found in the c-CRD). Impor-tantly, this disulfide was not observed to form when cellswere stressed with diamide. This group went on to showthat H2O2-induced but not diamide-triggered oxidation re-quired the participation of another protein called Gpx3 toproperly fold Yap1 (Delaunay et al. 2002). Gpx3 (aka Orp1)is one of several glutathione peroxidase homologs foundin S. cerevisiae (Inoue et al. 1999a). Unlike its homologsfor which evidence exists that these proteins act as bonafide glutathione peroxidases (Avery and Avery 2001), Gpx3has a primary role as a sensor protein and is covalentlylinked via its cysteine 36 to C598 of Yap1 upon H2O2 stress(Delaunay et al. 2002). This linkage is believed to stimulatethe inter-CRD disulfide bond formation between C303 andC598, a key modification required for the Yap1 response toH2O2.

While formation of the Gpx3 C36-C598 Yap1 disulfide isrequired for normal regulation of Yap1 by H2O2, it is notsufficient. Mutants lacking additional cysteines present ineither the n-CRD (C310) or the c-CRD (C629) exhibitedmajor defects in H2O2 resistance (Coleman et al. 1999;Delaunay et al. 2000). An explanation for these data wereprovided by solution of the structure of the H2O2-induceddisulfide-bonded form of Yap1 by NMR (Wood et al. 2004).This structure demonstrated the presence of a pair of disul-fide bonds linking C303-C598 and C310-C629. This struc-ture predicted that this dually disulfide-bonded form ofYap1 would be incapable of interacting with Crm1 as thebinding site for this export factor is occluded by the H2O2-induced structure of Yap1.





















































Along with Gpx3, a second protein required for H2O2-induced folding of Yap1 was found. This protein was desig-nated Ybp1 (Yap1-binding protein) and is required forin vivo folding of Yap1 in the presence of H2O2 but not di-amide (Veal et al. 2003; Gulshan et al. 2004). A homolog ofYbp1, called Ybp2, is also present in S. cerevisiae but itsfunction is presently unclear. Overproduction of Ybp2 (akaYbh1) can bypass a gpx3D mutant while overproduction ofYbp1 cannot (Gulshan et al. 2004). More recent work foundthat Ybp2/Ybh1 can associate with the kinetochore (Ohkuniet al. 2008) but its precise role in either oxidative stress orchromosome segregation remains unclear. Studies usinga mutant allele of YBP1 often found in laboratory yeaststrains called ybp1-1 (Veal et al. 2003; Okazaki et al.2005) demonstrated that the peroxiredoxin Tsa1 is capableof folding Yap1, albeit less effectively than the Ybp1/Gpx3system (Tachibana et al. 2009). It is interesting to note thatin vitro folding experiments have provided evidence thatYap1 in the presence of Gpx3 alone can properly fold atsome level (Okazaki et al. 2007). The role of Ybp1 mightbe to increase the efficiency of this process in vivo to morerapidly mount a defensive response to H2O2 challenge. Amodel summarizing our current picture of Yap1 traffickingduring oxidative stress is shown in Figure 6.

Yap1 homologs and redox stress: S. cerevisiae containsa family of proteins sharing sequence similarity with Yap1.These have been collectively referred to as the Yap family(Fernandes et al. 1997). While many of these factors havebeen well documented to play a role in stress responses (see(Rodrigues-Pousada et al. 2010 for a recent review), onlyCad1/Yap2 has been implicated in tolerance to oxidativestress. Cad1 exhibits the highest degree of sequence conser-vation in two segments of its protein chain. The amino-terminal DNA binding domain is similar to that of Yap1,and the c-CRD region of Yap1 is also closely conserved withCad1. Direct examination of the similarity of these domainsin Yap1 and Cad1 suggested that these c-CRD domains sharesome degree of functional equivalency in the response tocadmium but not to H2O2 (Azevedo et al. 2007).

Interestingly, the alkylhydroperoxidase protein Ahp1 hasbeen found to regulate the oxidative folding of Cad1 ina manner highly reminiscent of Yap1 and Gpx3 (Iwai et al.2010). Biochemical experiments indicate that Ahp1 formsa covalent intermediate with Cad1 in the c-CRD regionand catalyzes disulfide bond formation there. This foldingreaction is less well characterized than that of Yap1 butapparently involves formation of Ahp1–Cad1 heterodimersfollowed by Cad1 disulfide-bonded homodimers.

Although good evidence for the Ahp1-catalyzed Cad1folding exists, the functional contribution of Cad1 to oxida-tive stress phenotypes is minor (Iwai et al. 2010). In theabsence of the YAP1 gene, the role of Cad1 is more evident.Cad1 is absent from other species such as C. glabrata (datanot shown), suggesting that the presence of this factor maybe a specialized adaptation of S. cerevisiae. Overproduction

of Cad1 has major phenotypic effects on a variety of drugs towhich Yap1 also elicits tolerance (Bossier et al. 1993; Wuet al. 1993; Hirata et al. 1994). Oxidant resistance conferredby Cad1 does not appear to be the primary role of thistranscription factor.

Skn7: Skn7 was originally isolated as a high-copy numbersuppressor of a strain defective in b-glucan synthesis (Brownet al. 1993). The identification of this gene piqued extensiveinterest in its study as Skn7 contained protein elementsreminiscent of bacterial two-component regulatory systemsas well as a DNA binding domain related to Hsf1 (Brownet al. 1993). Later experiments indicated that Skn7 acteddownstream of a histidine kinase involved in the osmoticstress response called Sln1 (Li et al. 1998). Skn7 was linkedto oxidative stress tolerance by a genetic screen searchingfor mutations that cause sensitivity to peroxide (Krems et al.1996). One of these genes, originally called POS9, wasfound to be allelic with SKN7. Mutant strains lacking bothYap1 and Skn7 were no more sensitive to H2O2 than eithersingle mutant (Krems et al. 1996; Morgan et al. 1997), sug-gesting that these two transcriptional regulators act in thesame genetic pathway.

A likely explanation for this convergence of Yap1 andSkn7 function came from an analysis of transcriptional acti-vation by these factors on promoters involved in thioredoxinhomeostasis. S. cerevisiae contains three different genesencoding thioredoxins, an important antioxidant (Gan1991; Muller 1991; Pedrajas et al. 1999). Transcriptional

Figure 6 Yap1 folding and trafficking. A scheme for oxidant-specificfolding and nuclear import of Yap1 is shown. The four key regulatorycysteine residues in their reduced conditions are indicated by the purplecircles. Reduced Yap1 is imported into the nucleus at a basal rate butinteracts with the exportin Crm1 and is returned to the cytoplasm in theabsence of oxidative stress. In the presence of oxidants that directly act onYap1 (like diamide), C-terminal cysteine residues are oxidized or modified(yellow circle) in a manner that prevents Crm1 from recognizing thenuclear export signal (blue triangle) in the Yap1 C terminus. Yap1 accu-mulates in the nucleus and activates gene expression. Finally, duringchallenge by peroxides that engage the Gpx3/Ybp1 folding pathway,Gpx3 is covalently linked to cysteine 598 of Yap1 by a disulfide bond(linked red circles). This modification, along with the participation ofYbp1, catalyzes an intramolecular folding reaction that leads to a duallydisulfide bonded Yap1 form. This species also accumulates in the nucleusand can activate expression of genes required for the response to perox-ide stress.































































activation of the thioredoxin-encoding TRX2 gene by Yap1 isrequired for H2O2 resistance (Kuge and Jones 1994). Simi-larly, TRX2 is also a target for Skn7 regulation and loss ofeither Yap1 or Skn7 alone is sufficient to prevent H2O2 in-duction of TRX2 transcription (Morgan et al. 1997). A simpleexplanation for these data are that both Yap1 and Skn7 arerequired for H2O2-stimulated TRX2 expression. This modelwas directly supported by demonstration that both Yap1 andSkn7 bound to the TRX2 promoter at different sites (Morganet al. 1997). Importantly, loss of the aspartate residue thatserves as the ultimate acceptor for Sln1-mediated phosphor-ylation had no effect on Skn7-mediated oxidative stress re-sistance although this same mutation eliminated osmoticstress tolerance (Morgan et al. 1997; Ketela et al. 1998; Liet al. 1998).

The theme of Yap1 and Skn7 acting at a common pro-moter to induce oxidative stress tolerance is not restricted togenes influencing thioredoxin resistance. Global proteomicanalyses demonstrated that Yap1 controlled the expressionof a minimum of 32 different polypeptide chains (Lee et al.1999a). Fifteen of these factors required the presence ofboth Yap1 and Skn7 to be induced by H2O2. The cognatetarget genes for these proteins include the two dismutaseloci in S. cerevisiae (SOD1 and SOD2), a peroxiredoxin(TSA1), and an alkyl hydroperoxide reductase (AHP1),among others. One caveat to these data are the lack of directdemonstration for an involvement of Yap1 in their regula-tion. A large number of genes are also regulated by Yap1 ina Skn7 independent fashion. An example of such a locus isargued to be GSH1 encoding the g-glutamylcysteine synthe-tase enzyme, the rate-limiting step in glutathione biosynthe-sis (Ohtake and Yabuuchi 1991). The case for lack of GSH1regulation by Skn7 is difficult to make since the data arenegative. However, while the Yap1 control of GSH1 is clear(Wu and Moye-Rowley 1994), the response of GSH1 to Skn7has not been reported (Dormer et al. 2002).

While much is known of the molecular basis of Yap1regulation by oxidative stress, disappointingly little informa-tion is available detailing the control of Skn7 by oxidants.Skn7 is a constitutive nuclear protein and no evidence hasbeen obtained documenting any changes in the expressionof this factor in the presence of oxidants (Raitt et al. 2000).An intriguing observation linking Skn7 function with that ofthe heat shock transcription factor Hsf1 suggests a possiblemeans of modulating Skn7 transcriptional activity (Raittet al. 2000). Since Skn7 contains a DNA binding domainsimilar to that of Hsf1, cells lacking SKN7 were tested forthe ability to tolerate an acute heat shock. The skn7D strainwas found to be more sensitive than an isogenic wild-typestrain to this heat stress. Loss of Skn7 was also found toprevent the H2O2 induction of expression of the SSA1 gene,a locus encoding a major Hsp70 protein. Strikingly, Skn7was found to bind the Hsf1 recognition sequence as wellas associate with Hsf1 itself.

Together, these data suggest that Skn7 associates withHsf1 to mediate the H2O2-dependent induction of SSA1

gene expression. Skn7 has also been argued to associatewith an important transcriptional regulator of cell cycle pro-gression called Mbp1, although the linkage of this interac-tion and oxidative stress remains obscure (Machado et al.1997). The findings that two relatively unrelated transcrip-tion factors can both associate with Skn7 suggest that thismight be a means by which the activity of Skn7 could bemodulated at different promoters. Understanding the spec-trum of protein:protein interactions in which Skn7 partici-pates is an important future research question.

Several other features of Skn7 have been implicated incontributing to oxidative stress tolerance. Early work dem-onstrated that protein kinase A signaling was able to repressSkn7 function (Charizanis et al. 1999b). The mechanism ofthis inhibition is still uncertain. A genetic screen searchingfor factors required to support transcriptional activation ofa Gal4–Skn7 fusion protein identified two proteins that sat-isfied this criterion. The first, Ccp1, is a mitochondrial cyto-chrome c peroxidase and appears to be involved in signalingthe presence of mitochondrial oxidative stress to Skn7(Charizanis et al. 1999a). The second, known as Fap7, isan essential protein and contains a nucleotide triphospha-tase domain (Juhnke et al. 2000). Fap7 has also been impli-cated in ribosome subunit processing (Granneman et al.2005) and arsenite resistance (Takahashi et al. 2010). Fi-nally, recent data have defined phosphorylation sites in Skn7that appear to be associated with the onset of oxidativestress (He et al. 2009).

The unique role of Skn7 in the oxidative stress responsemakes the limited understanding of this factor a critical gapin our knowledge. Skn7 is of singular importance in H2O2

tolerance, which cannot be bypassed by overproduction ofYap1 or use of a variety of constitutively active forms of thistranscription factor (Coleman et al. 1999). One explanationfor the unique contribution of Skn7 to oxidative stress re-sistance gene transcription might come from its ability torecruit different mediator components to key target pro-moters. Suggestive data consistent with this model havecome from an analysis of the degradation of the transcrip-tion Mediator subunit cyclin C (also known as Srb11) (Kras-ley et al. 2006).

Cyclin C (CycC) is a subunit of the so-called Cdk8 sub-complex of the transcriptional Mediator (recently reviewedin Malik and Roeder 2010). Early evidence suggested pri-marily a negative role for the Cdk8 subcomplex (Holstegeet al. 1998; Kuchin and Carlson 1998; Elmlund et al. 2006).More recent work has provided strong support for the ideathat the Cdk8 subcomplex can also exert a positive influenceon gene expression (Cooper and Strich 2002). CycC is rap-idly degraded in response to oxidative stress and this deg-radation correlates with the induction of stress geneexpression (Cooper et al. 1999; Vincent et al. 2001). Useof CycC as bait in a two-hybrid assay detected interactionwith a protein referred to as Ask10 (activator of Skn7) (Pageet al. 1996), previously argued to be a positive regulator oftranscriptional activation by Skn7. Loss of ASK10 elicits an

















































































oxidant hypersensitive phenotype that can be suppressed byremoval of the CycC gene. Biochemical experiments demon-strate that CycC degradation is blocked in the absence ofAsk10 (Cohen et al. 2003).

A possible explanation of these data would come fromAsk10 and Skn7 interacting to remove the repressive effectof CycC from oxidant responsive promoters. Skn7 mightthen be able to exert an additional effect on Mediator, whichis required for induction of antioxidant gene expression.This central role of Mediator in permitting Skn7 functionwould fit well with what is known of the action of Yap1during H2O2 induction of gene expression. Properly foldedYap1 can interact with the Mediator subunit Rox3 and thisinteraction is crucial for normal TRX2 activation and H2O2

tolerance (Gulshan et al. 2005).Further data linking Skn7 with Mediator emerged from

a biochemical analysis of proteins associating with the Ccr4–Not complex, which is a global regulator of gene expressionwith a major role in stress resistance (Lenssen et al. 2002).This protein complex has been associated with activity of thegeneral stress transcriptional regulators Msn2/4 (Lenssenet al. 2005) (discussed in the following section) but alsointeracts with Skn7. Use of Not1 in a two-hybrid screendetected interaction with Skn7 (Lenssen et al. 2007). Skn7was also found to interact with another component of theNot module (Not5). Skn7:Not5 interaction was dependenton the presence of the Not4 protein, which is an E3 ubiquitinligase (Mulder et al. 2007). Deletion of the NOT4 gene led toincreased occupancy of Skn7 on two different oxidativestress-responsive promoters (but not to a third) and alsoappeared to modestly elevate H2O2 resistance. Finally, puri-fication of Skn7 detected the presence of the Cdk8 protein,a cyclin-dependent kinase associated with the Mediatorcomplex (Lenssen et al. 2007). The presence of Cdk8 wasfound to be required for the increased expression of a Skn7target gene (albeit a gene involved in osmotic stress resis-tance only) seen in a not4D strain.

These data support the view that Skn7 interacts witha range of general transcriptional regulators (Cdk8 andCcr4–Not complex) that are known to influence oxidativestress tolerance. These interactions may explain the uniquerole of Skn7 in regulation of the response to oxidative chal-lenge. Since these general transcription factors have notbeen demonstrated to interact with Yap1, their engagementby Skn7 could permit full oxidative stress response of rele-vant promoters. Solving the mechanistic puzzle of Skn7 in-put to oxidative stress resistance remains an important goal.

Msn2/4: Msn2 and Msn4 have already been consideredabove as these transcription factors are important partici-pants in heat shock tolerance. However, these factors alsoplay roles in resistance to oxidative stress. A mutant lackingboth MSN2 and MSN4 is highly sensitive to oxidative stress(Martinez-Pastor et al. 1996). This observation, coupledwith the large number of oxidative stress genes underMsn2/Msn4 control, suggests that these factors are respon-

sive to oxidant challenge. Recent experiments have directlyexamined the localization of Msn2/4 to the nucleus uponoxidative stress (Boisnard et al. 2009). Interestingly, im-portant differences with other stress-stimulated nuclearlocalization have been detected. First, PKA-dependentphosphorylation of Msn2/4 was not observed to change inresponse to H2O2 treatment. Second, the presence of theTrx1/2 thioredoxins was required for H2O2 to triggerMsn2/4 recruitment to the nucleus. A remarkable featureof H2O2-induced Msn2/4 nuclear localization is that onlya fraction (21%) of the cells exhibited accumulation of thesefactors in the nucleus while nuclear localization was seen for�100% of cells upon osmotic stress (Gorner et al. 1998;Boisnard et al. 2009). H2O2-dependent localization ofMsn2/4 could be improved by removal of the TRR1 geneencoding the thioredoxin reductase. This suggested themodel that oxidized Trx1/2 were required to cause Msn2/4 nuclear localization (Figure 1). However, redox control ofMsn2/4 localization does not appear to involve direct oxi-dative modification of Msn2/4 protein sequences as seen forthe cysteine residues in Yap1 (see above). The precise mech-anism of redox regulation of Msn2/4 function remains to bedetermined.

A potential complication in the analysis of oxidative stresscontrol of Msn2/4 is illustrated by consideration of the roleof a key target gene regulated by these factors. CTT1 wasone of the first genes demonstrated to be regulated byMsn2/4 and encodes the cytosolic catalase, an enzyme withobvious importance to the maintenance of redox balance.Strikingly, CTT1 disruption mutants are not sensitive toH2O2 in exponentially growing cells (Izawa et al. 1996).This behavior is in marked contrast to yap1D mutant strainsthat are exquisitely sensitive to H2O2 challenge in log-phasecultures (Schnell et al. 1992; Kuge and Jones 1994). How-ever, when late log-phase cultures are evaluated, this de-pendence is inverted (Izawa et al. 1996). Possibly, nuclearlocalization of Msn2/4 upon H2O2 stress is similarly depen-dent on growth phase of the cells. The most thorough studyof Msn2/4 nuclear recruitment during H2O2 stress was donewith log-phase cells, which leaves the tantalizing possibilitythat this process would be much more efficient in cells thathave undergone the diauxic shift into late log growth.

A final feature of Msn2/4 regulation that seems likely tobe of importance to the function of these factors duringoxidative stress is their regulated degradation by the protea-some upon stress treatment. Ubiquitination of Msn2 wasfound to be triggered by phosphorylation mediated by thecyclin-dependent kinase Cdk8 (also known as Srb10) (Chiet al. 2001). Later experiments demonstrated that Msn2 wasrapidly degraded when localized to the nucleus during heatshock and that this turnover required the function of theproteasome (Durchschlag et al. 2004; Lallet et al. 2004; Boseet al. 2005). These latter experiments did not investigate thestability of Msn2/4 after oxidative challenge but since bothheat or oxidative stress elevates nuclear levels of these fac-tors, it seems reasonable that Cdk8 may be involved in the


























































































degradation of these transcription factors under either stresscondition.

Antioxidant defenses

ROS are continuously produced in actively metabolizingcells. However, S. cerevisiae, like all organisms, contains ef-fective antioxidant defense mechanisms, which detoxifyROS as they are generated and maintain the intracellularredox environment in a reduced state. An oxidative stress issaid to occur when ROS overwhelm these defenses, result-ing in genetic degeneration and physiological dysfunction,leading eventually to cell death (Figure 5). Antioxidantdefenses include a number of protective enzymes that arepresent in different subcellular compartments and can beupregulated in response to ROS exposure (Table 3). Non-enzymic defenses typically consist of small molecules thatcan act as free radical scavengers; to date, only ascorbic acidand GSH have been extensively characterized in yeast.

Catalases: Catalases are ubiquitous heme-containing en-zymes that catalyze the dismutation of H2O2 into H2O andO2 (Figure 4). Yeast has two such enzymes: the peroxiso-somal catalase A, encoded by CTA1, and the cytosolic cat-alase T, encoded by CTT1. CTA1 expression is coordinatedwith peroxisomal fatty acid metabolism, suggesting thatCta1 may function in the detoxification of H2O2 generatedfrom fatty acid b-oxidation (Hiltunen et al. 2003). Ctt1 isthought to play a more general role as an antioxidant dur-ing exposure to oxidative stress, since CTT1 expression is in-duced by various stress conditions including heat, osmotic,starvation, and hydrogen peroxide stress (Martinez-Pastoret al. 1996). Surprisingly however, yeast mutants lackingboth catalases are unaffected in hydrogen peroxide toler-ance during exponential phase growth (Izawa et al. 1996).Redundancy in antioxidant defenses may account for thisapparently minor role in hydrogen peroxide tolerance,since loss of catalases exacerbates the peroxide sensitivityof glutathione mutants and the glutathione system is hy-persensitive to hydrogen peroxide in catalase mutants(Grant et al. 1998). However, catalases are important forthe acquisition of peroxide resistance following pretreat-ment with low doses of H2O2 and upon entry into station-ary phase, indicating a role during adaptive responses(Izawa et al. 1996).

Superoxide dismutases: Superoxide dismutases (SODs)convert the superoxide anion to hydrogen peroxide, whichcan then be reduced to water by catalases or peroxidases(Figure 4). SODs are ubiquitous antioxidants, which differ intheir intracellular location and metal cofactor requirementsbetween different organisms. Enzyme activity is dependenton redox cycling of the bound metal cofactor. Yeast containsa cytoplasmic Cu,Zn-SOD (Sod1) and a mitochondrial matrixMn-SOD (Sod2), which appear to play distinct roles duringoxidative stress conditions (Culotta et al. 2006). Cells deletedfor SOD1 are hypersensitive to superoxide-generating agents

such as paraquat and display a number of oxidative stress-related phenotypes including vacuole damage and increasedfree iron concentrations (Culotta 2000). Additionally, aero-bic inactivation of [4Fe-4S] cluster enzymes in sod1 mutantsresults in auxotrophies for methionine and leucine (Slekaret al. 1996; Wallace et al. 2004). While Sod1 is predomi-nantly cytosolic, it also localizes to the mitochondrial innermembrane space where it is thought to function in the de-toxification of superoxide generated from respiration (Sturtzet al. 2001). Mutants deleted for SOD2 are less affected ingrowth and stress sensitivity compared with sod1 mutantsbut do show a reduced ability to grow under respiratoryconditions (Van Loon et al. 1986). Sod2 is particularly re-quired during stationary phase growth, which may be linkedto superoxide generation from mitochondrial respiration(Longo et al. 1996). Cells deleted for SOD2 can grow underconditions of hyperoxia as a result of mutations that disruptthe mitochondrial electron transport chain, confirming therole of respiration in ROS production in yeast (Guidotet al. 1993). Similarly, the viability of sod1 sod2 mutantsduring long-term stationary phase incubation can be re-stored by similar mutations, further implicating the mito-chondrial electron transport chain as the major source ofROS in yeast (Longo et al. 1996).

Methionine sulfoxide reductase: Amino acids are susceptibleto oxidation by ROS (Stadtman and Levine 2003). Methionineresidues are particularly susceptible, forming a racemic mix-ture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO) in cells (Dean et al. 1997). Mostorganisms contain methionine sulfoxide reductases (MSRs),which protect against methionine oxidation by catalyzingthiol-dependent reduction of oxidized Met residues. This isparticularly important because it means that methionine ox-idation is readily reversible and can play an antioxidant rolein scavenging ROS (Stadtman et al. 2003). Yeast containsthree MSR enzymes that are required for resistance againstoxidative stress (fRMsr/MsrA/MsrB) (Le et al. 2009). fRMsris thought to be the main enzyme responsible for the reduc-tion of free Met-R-SO, whereas MsrA and MsrB are activewith Met-S-SO and Met-R-SO in proteins. A triple fRMsr/MsrA/MsrB mutant is viable on media containing methio-nine, but cannot grow if methionine is substituted withMet-SO (Le et al. 2009).

Thioredoxins: S. cerevisiae, like most eukaryotes, containsa cytoplasmic thioredoxin system, which functions in pro-tection against oxidative stress (Figure 7). This comprisestwo thioredoxins (TRX1 and TRX2) and a thioredoxin re-ductase (TRR1) (Gan 1991). Thioredoxin mutants are auxo-trophic for sulfur amino acids, since thioredoxins are thesole hydrogen donors for PAPS reductase, the enzyme thatconverts 39-phosphoadenosine 59-phosphosulfate (PAPS) tosulfite (Muller 1991). Mutants deleted for TRX1 and TRX2are also affected in the cell cycle with a prolonged S phaseand shortened G1 interval (Muller 1991). This correlates

































with the role of cytoplasmic thioredoxins as the major reduc-tants of ribonucleotide reductase during S phase (Koc et al.2006; Camier et al. 2007). As in most organisms, yeast thi-oredoxins are active as antioxidants and play key roles inprotection against oxidative stress induced by various ROS(Kuge and Jones 1994; Izawa et al. 1999). A major part ofthe antioxidant function of thioredoxins is mediated by per-oxiredoxins (Prx’s, see below). Oxidized thioredoxins (Trx1/Trx2) are rapidly observed (,15 sec) following exposure tohydrogen peroxide and are detected for.1 hr before return-ing to the reduced form (Okazaki et al. 2007). Trx2 appearsto play the predominant role as an antioxidant, sincemutants lacking TRX2 are hypersensitive to hydroperoxidesand mutants containing TRX2, in the absence of TRX1, showwild-type resistance to oxidative stress (Garrido and Grant2002). However, Trx1 and Trx2 appear to be functionallyredundant as antioxidants. This is emphasized by the similarredox midpoint potentials (Em) of Trx1 and Trx2 (2275 and

2265 mV, respectively), indicating the interchangeable na-ture of these proteins (Mason et al. 2006). The differentialrequirement for Trx1 and Trx2 appears to be related todifferences in gene expression; TRX2 expression is stronglyupregulated in response to oxidative stress conditions,whereas TRX1 may serve an ancillary or back-up role duringconditions in which TRX2 is insufficient to provide an anti-oxidant defense (Garrido and Grant 2002).

Yeast also contains a complete mitochondrial thioredoxinsystem, comprising a thioredoxin (Trx3) and a thioredoxinreductase (Trr2) (Figure 7) (Pedrajas et al. 1999). The re-dox states of the cytoplasmic and mitochondrial thioredoxinsystems are independently maintained and cells can survivein the absence of both systems (Trotter and Grant 2005).The yeast mitochondrial thioredoxin system has been impli-cated in protection against oxidative stress generated duringrespiratory metabolism. However, the mitochondrial thiore-doxin reductase was found to have an antioxidant role

Table 3 Protective enzymes that can be up-regulated in response to ROS exposure

Antioxidant Gene Locationa Activity

The thioredoxin systemThioredoxin TRX1, TRX2 Cyt Disulfide oxidoreductase activity

TRX3 Mit Mitochondrial disulphide oxidoreductase activityThioredoxin reductase TRR1 Cyt Reduces oxidized thioredoxins (Trx1, Trx2)

TRR2 Mit Reduces oxidized thioredoxin (Trx3)Peroxiredoxin TSA1, TSA2 Cyt 2-Cys Prx, thioredoxin peroxidase and chaperone activity

AHP1 Cyt 2-Cys Prx, thioredoxin peroxidase particularly withalkyl hydroperoxides

DOT5 Nuc Nuclear 2-Cys Prx, functions in telomeric silencingPRX1 Mit Mitochondrial 1-Cys Prx, thioredoxin peroxidase activity

The glutathione systemGSH synthesis GSH1, GSH2 Cyt Catalyze two ATP-dependent steps in GSH biosynthesisGlutathione reductase GLR1 Cyt/Mit Recycles oxidized GSSG to reduced GSH, co-localizes to Cyt and MitGlutathione transferase GTT1 ER Catalyze the conjugation of GSH to various electrophiles

GTT2 MitGTO1 Per Omega class glutathione transferaseGTO2, GTO3 Cyt Omega class glutathione transferase

Glutathione peroxidase GPX1, GPX2 Cyt Phospholipid hydroperoxide glutathione peroxidaseGPX3 Cyt Phospholipid hydroperoxide glutathione peroxidase,

Yap1 signal transducerGlutaredoxin GRX1 Cyt Glutathione disulfide oxidoreductase activityGRX2 Cyt/Mit Glutathione disulfide

oxidoreductase activity,colocalizes to Cyt and Mit

GRX3, GRX4 Nuc Monothiol glutaredoxinGRX5 Mit Monothiol glutaredoxin, synthesis/assembly of iron-sulfur centersGRX6, GRX7 Gol Cis-Golgi localized monothiol glutaredoxinsGRX8 Cyt Glutathione disulfide oxidoreductase activity

Superoxide dismutase SOD1 Cyt/Nuc Catalyze the dismutation of superoxide intooxygen and hydrogen peroxide

SOD2 MitCatalase CTT1 Cyt Catalyze the reduction of hydrogen peroxide to water and oxygen

CTA1 PerMethionine sulphoxide MXR1 (MSRA) Cyt Catalyze thiol-dependent reduction of methionine (S)-sulfoxideReductase MXR2 (MSRB) Mit Catalyze thiol-dependent reduction of methionine (R)-sulfoxide

fRMsr (YKL069W) Cyt Catalyze thiol-dependent reduction of free Met-R-SOErythroascorbate ALO1 Mit D-arabinono-1,4-lactone oxidase, final step of

erythroascorbate synthesisa Location is based on information provided at http://www.yeastgenome.org/.



















http://www.yeastgenome.org/

independent of thioredoxin since mutants deleted for TRR2are sensitive to oxidative stress, compared with trx3 mutants,which are unaffected in oxidant resistance (Pedrajas et al.2000; Trotter and Grant 2005).

Peroxiredoxins: Peroxiredoxins (Prx) have multiple roles instress protection, acting as antioxidants, molecular chaper-ones, and in the regulation of signal transduction (Woodet al. 2003). They use redox-active Cys residues to reduceperoxides and have been divided into two classes, the 1-Cysand 2-Cys Prx’s, on the basis of the number of Cys residuesdirectly involved in catalysis. Typical 2-Cys Prx’s are activeas a dimer and contain two redox-active Cys residues thatare required for enzyme activity (Chae et al. 1994; Park et al.2000). During catalysis, the peroxidatic cysteine residue ofone subunit is oxidized to a sulfenic acid, which condenseswith the resolving cysteine from the other subunit to forma disulfide that is reduced by thioredoxin (Figure 7). Threecytoplasmic 2-Cys Prx’s (Tsa1, Tsa2, and Ahp1) have beendescribed in yeast (Morgan and Veal 2007). All three displaythioredoxin peroxidase activity, but appear to play distinctphysiological roles. Tsa1 has best been characterized as anantioxidant in the detoxification of hydroperoxides (Garridoand Grant 2002; Wong et al. 2004), but has also been shownto act as a chaperone that promotes resistance to heat andreductive stresses (Jang et al. 2004; Rand and Grant 2006).Tsa2 is highly homologous to Tsa1 and possesses similarperoxidase and chaperone activities, but is expressed at sig-nificantly lower levels than Tsa1 (Jang et al. 2004). Ahp1 isactive as an antioxidant, but in contrast to Tsa1, its catalyticefficiency is greater with alkyl hydroperoxides than withH2O2 (Lee et al. 1999b; Park et al. 2000). Differences inthe cytoplasmic Prx’s are further highlighted by the findingthat in contrast to Tsa1 and Tsa2, overoxidation of Ahp1 tocysteine–sulfinic acid is not reversed by Srx1 (Biteau et al.2003). Dot5 (nTPx) is a nuclear 2-Cys Prx, which is mostactive against alkyl hydroperoxides (Cha et al. 2003). How-ever, it has been proposed to play a minor role as an anti-oxidant, predominantly functioning in telomeric silencing(Izawa et al. 2004).

Yeast Prx1 is a member of the 1-Cys family of Prx’s and isactive as a peroxidase (Pedrajas et al. 2000). 1-Cys Prx’scontains a peroxidatic cysteine, but do not contain a resolv-ing cysteine residue. Since 1-Cys Prx’s cannot therefore forma disulfide, the cysteine–sulfenic acid generated by reactionwith peroxides is thought to be reduced by a thiol-contain-ing electron donor, but this reaction mechanism is poorlyunderstood. The peroxidatic cysteine residue of Prx1 is ox-idized to the sulfenic acid form by hydroperoxides (Gree-tham and Grant 2009). GSH efficiently attacks the sulfenicacid intermediate, resulting in the formation of glutathiony-lated Prx1 (Figure 7). Active Prx1 appears to be regeneratedby reduction by Trr2 (Greetham and Grant 2009), or alter-natively, by the Grx2 glutaredoxin (Pedrajas et al. 2010).This is an important finding since it suggests that there isa functional overlap between the GSH/glutaredoxin and

thioredoxin systems in mitochondrial antioxidant protec-tion. Further studies are required to decipher the molecularregulation of the different yeast Prx isoforms to better un-derstand the cellular functions of this family of proteins. Thisis important given the increasing evidence that human Prx’sare implicated in various disease processes (Immenschuhand Baumgart-Vogt 2005; Kang et al. 2006). Importantly,a strain lacking all five yeast Prx’s is viable but is hypersensi-tive to oxidative stress (Wong et al. 2004). It also displays anincreased rate of spontaneous mutations, indicating thatPrx’s function to maintain genome stability in the presenceof endogenous ROS.

The glutathione system: The oxidation of sulfhydryl groupsis one of the earliest observable events during ROS-mediated damage. This underlies the importance of GSH(g-glutamylcysteinylglycine) which is typically found as themost abundant low molecular-weight sulfhydryl compound(mM concentrations) in most organisms. Many roles havebeen proposed for GSH in a variety of cellular processes in-cluding amino acid transport; synthesis of nucleic acids andproteins; modulation of enzyme activity; and metabolism ofcarcinogens, xenobiotics, and ROS (Schafer and Buettner

Figure 7 Comparison of cytoplasmic and mitochondrial thioredoxin sys-tems. The yeast cytoplasmic thioredoxin system comprises two thioredox-ins (Trx1-2) and a thioredoxin reductase (Trr1). The oxidized disulphideform of thioredoxin is reduced directly by NADPH and thioredoxin reduc-tase (Trr1). The yeast cytosol contains three typical 2-Cys Prx’s (Tsa1, Tsa2,and Ahp1) but only Tsa1 is shown for simplicity. 2-Cys Prx’s are active asa dimer and contain two redox active Cys residues that are directly in-volved in enzyme activity. During reduction of hydroperoxides, a disul-phide bond is formed between the peroxidatic cysteine (SP) of onesubunit and the resolving cysteine (SR) from the other subunit of thedimer. This disulphide is reduced by thioredoxin. Yeast contains a com-plete mitochondrial thioredoxin system including a thioredoxin (Trx3) andthioredoxin reductase (Trr2). The substrate(s) of Trx3 is currently un-known. Mitochondria contain a single 1-Cys Prx (Prx1). The peroxidaticCys residue of Prx1 is oxidized to the sulphenic form by hydroperoxides.Oxidized Prx1 is glutathionylated and reduced by Grx2 or Trr2 to regen-erate the active enzyme. Reduced components are shown in blue.
























2001). Not surprisingly therefore, GSH is an essential me-tabolite in eukaryotes, and for example, mice that are de-ficient in GSH biosynthesis die rapidly (Shi et al. 2000).Similarly, GSH is an essential metabolite in yeast where itappears to be required as a reductant during normal growthconditions (Grant et al. 1996b). Oxidative stress convertsglutathione to its oxidized disulfide form (GSSG) (Figure8). However, glutathione is predominantly present in its re-duced GSH form in yeast and other eukaryotes due to theconstitutive action of glutathione reductase (Glr1). Glr1 isan NADPH-dependent oxidoreductase, which convertsGSSG to GSH using reducing power generated by the pen-tose phosphate pathway (López-Barea et al. 1990). YeastGLR1 is not essential for normal aerobic growth, but is re-quired for viability during exposure to oxidative stress andfollowing starvation conditions (Grant et al. 1996a,c).

GSH is synthesized via two ATP-dependent steps (Figure8). g-Glutamylcysteine synthetase (Gsh1) catalyzes the firstand rate-limiting step where the dipeptide g-Glu-Cys isformed from glutamate and cysteine (Lisowsky and Meister1993). The second step is catalyzed by glutathione synthe-tase (Gsh2), which ligates g-Glu-Cys with glycine (Grantet al. 1997). GSH biosynthesis is tightly regulated via twooverlapping mechanisms. Gsh1 enzyme activity is feedbackinhibited by GSH (Meister 1988) and GSH1 expression isregulated by the cellular concentrations of GSH in parallelwith sulfur amino acid biosynthesis (Wheeler et al. 2002,2003). Mutants deleted for GSH1 are unable to grow inthe absence of exogenous GSH, but can undergo a limitednumber of cell divisions in the absence of GSH during whichthey utilize preaccumulated stores of GSH (Lee et al. 2001;Spector et al. 2001). This has enabled the gsh1 mutant to beextensively used in studies aimed at determining the role ofGSH during oxidative stress conditions, by examining thestress sensitivity of the gsh1 mutant in the absence of exog-enous GSH. These studies have shown that mutants lackingGSH1 are generally sensitive to oxidants including perox-ides, the superoxide anion, lipid hydroperoxides and theirbreakdown products, and heavy metals (Grant et al. 1996b;Evans et al. 1997; Turton et al. 1997; Thorsen et al. 2009).

Despite the apparent importance of GSH in protectionagainst ROS, this may not explain the essential function ofGSH, since the lethality of a gsh1 mutant grown in the ab-sence of GSH cannot be rescued by anaerobic growth con-ditions (Spector et al. 2001). GSH is important, however,during stress conditions in its role as a cofactor for stressdefense enzymes, including glutathione transferase (GSTs)and glutathione peroxidases (Gpx’s) as described below(Figure 8). The gsh1 mutant has also been used to modelthe effects of GSH depletion, which can be caused by severalprocesses, including conjugation to xenobiotics, excretion,and decreased synthesis and has been implicated in degen-erative diseases, cell aging, and apoptosis (Jain et al. 1991;Hardings et al. 1997). GSH depletion in yeast causes a G1cell cycle arrest and hence its essential function is unlikely tobe associated with a lack of ribonucleotide reductase activity,

which would be expected to affect the S phase of the cellcycle (Spector et al. 2001). The eukaryotic requirement forGSH is still uncertain but it may be explained due to its role inthe synthesis of [4Fe-4S] clusters, which are essential forviability (Toledano et al. 2007).

Glutaredoxins: Glutaredoxins (Grx) are small heat-stableoxidoreductases, which were first discovered in E. coli asGSH-dependent hydrogen donors for ribonucleotide reduc-tase (Holmgren 1989). Classical cellular glutaredoxins con-tain a conserved dithiol active site (Cys-Pro-Tyr-Cys) andform part of the glutaredoxin system, in which glutathionereductase transfers electrons from NADPH to glutaredoxinsvia GSH. They have proposed roles in many cellular pro-cesses including protein folding and regulation, reductionof dehydroascorbate, and protection against ROS and sulfurmetabolism (Holmgren 1989). Two yeast genes encode clas-sical dithiol glutaredoxins (GRX1 and GRX2) (Luikenhuiset al. 1997). Grx1 and Grx2 are active as GSH-dependentoxidoreductases, but appear to have distinct cellular func-tions. Strains deleted for GRX1 are sensitive to oxidativestress induced by the superoxide anion, whereas strainslacking GRX2 are sensitive to hydrogen peroxide. This dif-ference in oxidant sensitivity may reflect differences in thesubstrate proteins regulated by Grx1 and Grx2, or in theirability to detoxify ROS-mediated damage (Luikenhuis et al.1997). Differences in the expression of GRX1 and GRX2 havealso been described further indicating that the two glutare-doxin isoforms may play distinct roles during normal growthand stress conditions (Grant et al. 2000). Both genes areregulated in response to oxidative stress conditions viastress-responsive STRE elements, although the induction ofGRX2 is much more rapid and stronger compared with GRX1.

Figure 8 The glutathione system. GSH is synthesized from its constitu-ent amino acids via two ATP-dependent steps. In the first step, Gsh1(g-glutamylcysteine synthetase) catalyses the formation of the dipeptideg-glutamylcysteine (g-Glu-Cys) from glutamic acid and cysteine. In thesecond step, Gsh2 (glutathione synthetase) catalyses the ligation ofg-Glu-Cys with glycine. GSH can be oxidized to GSSG by ROS or inreactions catalyzed by Grx1–8 and Gto1–3. Reduced GSH is regener-ated in an NADPH-dependent reaction catalyzed by Glr1 (glutathionereductase). GSH can be conjugated to xenobiotics (RX) by GSTs, includ-ing Gtt1–2 and Grx1–2. GSH conjugates are transported to the vacuoleby the Ycf1 GS-X pump.



























Six related glutaredoxins have also now been identifiedin yeast (Grx3–8). They are found in different subcellularcompartments including nuclear (Grx3/4), the mitochondrialmatrix (Grx5), and the early secretory pathway (Grx6/7)(Rodriguez-Manzaneque et al. 1999; Molina et al. 2004;Izquierdo et al. 2008; Mesecke et al. 2008; Eckers et al. 2009).Grx3/4 play an essential role in intracellular iron trafficking(Muhlenhoff et al. 2010) and Grx5 is required for mitochon-drial [4Fe-4S] cluster assembly (Rodriguez-Manzanequeet al. 2002). Grx3–5 differ from dithiol glutaredoxins in thatthey contain a single cysteine residue at their putative activesites. They are important during the oxidative stress re-sponse since they function to regulate iron metabolismand availability. Grx6 and Grx7 are also monothiol glutare-doxins, which are thought to function in sulfhydryl regula-tion in the early secretory pathway during stress conditions(Izquierdo et al. 2008; Mesecke et al. 2008). Grx8 is a dithiolglutaredoxin but is not thought to function in the oxidativestress response (Eckers et al. 2009).

Glutathione peroxidases: Eukaryotic glutathione peroxi-dases (Gpx’s) are thought to provide the major enzymaticdefense against oxidative stress caused by hydroperoxides.They reduce hydrogen peroxide and other organic hydro-peroxides, such as fatty acid hydroperoxides, to the corre-sponding alcohol, using reducing power provided by GSH(Michiels et al. 1994). Mammalian cells also contain phos-pholipid hydroperoxide Gpx’s (PHGpx’s), which are able toreduce membrane phospholipid hydroperoxides (Roveriet al. 1994). Interestingly, yeast does not contain any classi-cal Gpx’s, but expresses three PHGpx’s encoded by GPX1-3(Inoue et al. 1999b; Avery and Avery 2001). These PHGpxenzymes have activity with phospholipid hydroperoxides aswell as nonphospholipid hydroperoxides and are able toprotect membrane lipids against peroxidation. Yeastmutants lacking PHGpx’s are hypersensitive to hydroperox-ides, including phospholipid hydroperoxides with Gpx3appearing to account for the majority of activity (Averyand Avery 2001; Avery et al. 2004). However, the role ofGpx3 in oxidant tolerance is complicated, given its addi-tional function as a peroxide sensor and activator of Yap1(Delaunay et al. 2002). Furthermore, subsequent studieshave shown that yeast PHGpx’s are better classified as atyp-ical 2-Cys peroxiredoxins, since they form an intramoleculardisulfide bond as part of their catalytic cycle, which iscleaved by thioredoxin (Delaunay et al. 2002; Tanakaet al. 2005; Ohdate et al. 2010).

Glutathione transferases: Glutathione transferases (GSTs)are a major family of proteins, which are involved in thedetoxification of many xenobiotic compounds (Sheehanet al. 2001). They catalyze the conjugation of electrophillicsubstrates to GSH prior to their removal from cells via glu-tathione conjugate pumps (Figure 8). Two genes encodingfunctional GSTs, designated GTT1 and GTT2, have beenidentified in yeast (Choi et al. 1998). Purified recombinant

Gtt1 and Gtt2 are active in a GST assay using 1-chloro-2,4-dinitrobenzene (CDNB) as a model substrate, but share lim-ited sequence homology with each other and with GSTsfrom other species. Strains lacking GTT1 and GTT2 are via-ble and are unaffected in growth during normal aerobicconditions. In addition, the gtt1Δ gtt2Δ mutant does notshow increased sensitivity to CDNB, which is surprisinggiven that Gtt1 and Gtt2 would be expected to detoxifyCDNB via conjugation (Choi et al. 1998) and the Ycf1 GS-X pump is required for CDNB resistance (Li et al. 1996). Thismay be explained by functional redundancy with glutare-doxins, since yeast glutaredoxins (Grx1 and Grx2) are activeas GSTs with substrates such as CDNB (Collinson and Grant2003). Mutant analysis has confirmed that Grx1 and Grx2have an overlapping function with Gtt1 and Gtt2, sincesimultaneous loss of all four genes substantially reduces cel-lular GST activity and causes sensitivity to stress conditions,including exposure to xenobiotics, heat, and oxidants (Col-linson et al. 2002; Collinson and Grant 2003). S. cerevisiaealso contains three omega class GSTs encoded by GTO1,GTO2, and GTO3 (Barreto et al. 2006; Garcera et al.2006). These enzymes are induced in response oxidants un-der the control of Yap1 and STRE-responsive elements.However, Gto1-3 are not active as GSTs with CDNB, butshow activity as thiol transferases (glutaredoxins) and asGpx’s. Mutants lacking GTO1-3 display sensitivity to hydro-peroxides and thiol oxidants and also show additive effectswith gtt mutants including strong sensitivity to cadmium(Barreto et al. 2006; Garcera et al. 2006). The requirementfor GSTs in S. cerevisiae appears complex and is provided byredundant gene families. Part of this redundancy may beexplained by the differing locations of GSTs, including cyto-solic (Grx1, Grx2, Gto2, and Gto3), ER (Gtt1), peroxisomal(Gto1), and mitochondrial (Gtt2 and Grx2).

Ascorbic acid: Ascorbic acid is a water soluble antioxidant,which commonly acts in a redox couple with glutathione inmany eukaryotes (Winkler et al. 1994). However, the rele-vance of ascorbate to the yeast oxidative stress response isunclear since yeast contains a 5-carbon analog, erythroascor-bate, which may have limited importance as an antioxidant.Strains deleted for ALO1, encoding D-arabinono-1,4-lactoneoxidase, which catalyzes the final step in erythroascorbatebiosynthesis, are sensitive to hydrogen peroxide and thesuperoxide anion (Huh et al. 1998). However, the extremelylow levels of erythroascorbate detected in yeast make itsfunctional role as an antioxidant questionable (Spickettet al. 2000). It has also been suggested that ascorbate mayact as the physiological reductant for 1-Cys Prx’s (Monteiroet al. 2007), although there does not appear to be anin vivo requirement for erythroascorbate to maintain theyeast 1-Cys peroxiredoxin (Greetham and Grant 2009).

Cellular responses to ROS

Yeast cells respond to ROS by altering the expression ofgenes encoding antioxidant defense mechanisms and genes
























































encoding enzymes, which repair and detoxify the resultingcellular damage. This forms the basis for inducible adaptiveresponses, where for example, cells treated with a low doseof oxidant can adapt to become resistant to a subsequentand otherwise lethal treatment. Adaptive responses havebeen extensively studied in S. cerevisiae, where it is now wellestablished that there are a number of cellular responsesthat ensure the survival of the cell following exposure tooxidants such as hydrogen peroxide, or products of oxida-tive damage (Collinson and Dawes 1992; Jamieson 1992;Turton et al. 1997). While there has been considerable re-search on the cellular antioxidant defense systems, whichprotect against ROS, less is known about how adaptationoccurs. Its nature depends on the treatment. For example,heat shock induces resistance to hydrogen peroxide, buthydrogen peroxide does not have the reverse effect. Cellsadapted to hydrogen peroxide treatment become resistant tomenadione (a superoxide generator), but not vice versa(Flattery-O’Brien et al. 1993). This hierarchical response tostress may indicate the existence of a number of differentadaptation systems, which have overlapping components(Temple et al. 2005). De novo protein synthesis is knownto be required for the induction of resistance to ROS, sinceit is abrogated by treatment with translation inhibitors suchas cycloheximide (Collinson and Dawes 1992). Adaptationmay therefore be explained by the pattern of proteins thatare produced in response to different oxidant treatments. Inagreement with this idea, extensive transcriptional andtranslational reprogramming is evident during adaptivetreatments (Godon et al. 1998; Gasch et al. 2000; Shentonet al. 2006). It is also clear that yeast cells undergo extensivemolecular changes during oxidant exposure that are sum-marized in the following sections.

Redox homeostasis and oxidative stress: Cells must re-spond to an oxidative stress by regulating their thiol systemsto maintain the redox balance of the cell. There is alsoincreasing evidence that ROS play important regulatoryfunctions in cell signaling and thiol groups are key media-tors of oxidative signal transduction (Rhee et al. 2005).However, there is a limited understanding at present ofhow cells protect against the deleterious consequences ofROS, while allowing their role in cell signaling (D’Autréauxand Toledano 2007; Veal et al. 2007). The composition andredox state of available thiols in protein and low molecularweight compounds is highly dynamic and varies dependingon the growth conditions and local environment. Signaltransduction pathways generally involve specific protein–protein interactions and increasing evidence indicates thatoxidizing agents can control these interactions to bringabout controlled signaling events. Cysteine residues areamong the most easily oxidized residues in proteins, andmost ROS-specific regulatory mechanisms are based on theoxidation of protein-cysteine residues. This is primarily dueto the high reactivity of Cys residues and the ability of cys-teine residues to cycle between different oxidation states (e.g.,

disulfide, sulfenic acid, sulfinic acid, and sulfonic acid). Animportant precedent is provided by the Yap1 transcriptionfactor, where Prx’s act as peroxide sensors and activators.Cys residues react relatively slowly with ROS, but reactivitycan be significantly enhanced by their ionization state, whichis dependent on the local protein environment. This is em-phasized by the finding that H2O2 stress in yeast causes pro-tein-specific oxidation rather than general nonspecificoxidation of protein-SH groups (Le Moan et al. 2006).

All organisms contain complex regulatory machinery tomaintain their redox homeostasis, primarily regulated by theGSH/glutaredoxin and thioredoxin systems. These systemsare thermodynamically linked, since each uses NADPH asa source of reducing equivalents (Holmgren 1989). Exten-sive overlaps between the thioredoxin and GSH/glutare-doxin systems have been identified in yeast. For example,GLR1 was identified in a genetic screen for mutations thatconfer a requirement for thioredoxins, providing the firstin vivo evidence that thioredoxins and the GSH system havean overlapping redox function (Muller 1996). Subsequently,loss of TRX1 and TRX2 was shown to cause compensatorychanges in glutathione levels and redox state confirming thetight regulatory link between the thioredoxin system andglutathione (Muller 1996; Garrido and Grant 2002). Exten-sive deletion analyses in yeast have further identified con-siderable genetic redundancy in these systems (Trotter andGrant 2003; Toledano et al. 2007; López-Mirabal andWinther 2008). The thioredoxin system has also been shownto function as an alternative reduction system for GSSG,although the physiological significance of this reduction re-action is not yet clear (Tan et al. 2010). The reason(s) forthis apparent functional redundancy in highly conserved re-dox systems remains an important unanswered question.Extensive biochemical analyses suggest that the thioredoxinsystem, and not the glutathione system, is the predominantantioxidant system in yeast, since the main peroxidaseenzymes including peroxiredoxins (Tsa1, Tsa2, Ahp1, andDot5) and glutathione peroxidase-like enzymes (Gpx1-3) allappear to depend on the thioredoxin system for reduction(Toledano et al. 2007). However, one exception to this ruleis now known, since mitochondrial Prx1 reduction requiresglutathione (Greetham and Grant 2009).

Glutathione can form mixed disulfides with protein-SHgroups (GSSP) in response to ROS exposure. Glutathiony-lation is a reversible post-translational modification, whichis particularly important since it can both protect cysteineresidues from irreversible oxidation and can regulate theactivity of many target proteins. Eukaryotic glutaredoxinshave long been thought to protect against oxidative stress bycatalyzing the reduction of protein mixed disulfides withGSH (Cotgreave and Gerdes 1998). GSSP levels in yeast aremaintained at low levels, but are increased following expo-sure to hydrogen peroxide (Grant et al. 1998). Additionally,GSSP levels are regulated in parallel with the growth cycleand are maximal during stationary phase growth (Greethamet al. 2010). However, unlike most eukaryotes examined to












date, thioredoxins, and not glutaredoxins, appear to be themajor deglutathionylases in yeast, providing further evi-dence that the thioredoxin system and glutathione have ex-tensive overlapping redox functions (Greetham et al. 2010).This does however leave open the question as to the func-tion of yeast glutaredoxins. As mentioned previously, Grx1and Grx2 are active with ribonucleotide reductase, but noother substrates have been confirmed in vivo. Glutaredoxinshave been shown to reduce arsenate reductase (Acr2)in vitro, but it is unclear whether this is a physiological re-action (Mukhopadhyay et al. 2000). This is in contrast toplants such as Arabidopsis, where for example .100 puta-tive redoxin targets have been identified (Meyer et al.2008). Glutaredoxins are the ultimate electron donors forthe glutathione system, but at present we have only a limitedknowledge of their physiological targets in yeast. This is nota trivial problem since the identity and strength of any targetinteractions can change during growth conditions, whichalter the cellular redox balance.

Translational regulation of gene expression: Global in-hibition of protein synthesis is a common response to stressconditions. However, it is becoming increasingly recognizedthat differential translational control of specific mRNAs isrequired for survival during growth under stress conditions(Preiss et al. 2003; Smirnova et al. 2005). Inhibiting proteinsynthesis during oxidative stress conditions may preventcontinued gene expression during potentially error-proneconditions as well as allow for the turnover of existingmRNAs and proteins while gene expression is reprog-rammed to deal with the stress. Yeast cells respond to hy-drogen peroxide stress with a rapid and reversible inhibitionof protein synthesis with low adaptive concentrations ofhydrogen peroxide inhibiting translation by .60% within15 min (Godon et al. 1998; Shenton and Grant 2003; Shen-ton et al. 2006). The level of inhibition is dose dependentand is largely mediated by the Gcn2 protein kinase thatphosphorylates the a-subunit of translation initiation factor2 (eIF2) (Shenton et al. 2006). eIF2 is a guanine nucleotidebinding factor, which in its GTP-bound form interacts withthe initiator methionyl-tRNA (Met-tRNAi

Met) to form a ter-nary complex that is competent for translation initiation.eIF2 is released from the ribosome as a binary complexwith GDP, which is removed and replaced by GTP in aguanine-nucleotide exchange reaction catalyzed by eIF2B.Met-tRNAi

Met can only bind the eIF2/GTP complex, so trans-lational control can be regulated by the activity of eIF2B. Inyeast and mammalian cells, this is achieved by phosphory-lation of eIF2a at a conserved serine (Ser51) residue (Pavittet al. 1998; Harding et al. 2000). Phosphorylation convertseIF2 from a substrate to a competitive inhibitor of eIF2B andthe resulting decrease in eIF2B activity leads to reducedternary complex levels, which inhibits translation initiation(Figure 9) (Pavitt et al. 1998).

Gcn2 was first characterized for its activation in responseto amino acid starvation (Hinnebusch 2005). Depletion of

amino acids leads to an accumulation of uncharged tRNA,which activates Gcn2 via its HisRS-related domain. It is nowknown that Gcn2 can be activated in response to a variety ofconditions including nutrient starvation (amino acids,purines, glucose) and exposure to sodium chloride, rapamy-cin, ethanol, and volatile anesthetics (Hinnebusch 2005;Palmer et al. 2005). It is likely that these other stress con-ditions also ultimately affect the levels of uncharged tRNA inthe cell. For example, volatile anesthetics inhibit amino aciduptake (Palmer et al. 2005) and Gcn2 is activated by glucosestarvation partly through an effect on vacuolar amino acidpools (Yang et al. 2000). Phosphorylation of eIF2a appearsto be a general response to ROS, since Gcn2 can be activatedby exposure to an organic hydroperoxide (cumene hydro-peroxide), a thiol oxidant (diamide), and a heavy metal(cadmium) (Mascarenhas et al. 2008). Oxidative stress con-ditions appear to activate Gcn2 via an effect on unchargedtRNA levels since mutations in the HisRS-like domain abol-ish Gcn2-inhibition (Mascarenhas et al. 2008). The pleiotro-pic nature of oxidative stress means that it is currently notknown how ROS cause an amino acid starvation and/oreffect cellular uncharged tRNA levels. ROS may conceivablyaffect the transport and storage of amino acids withincells, similar to the effect of glucose starvation. Additionally,proteins and nucleic acids, which are required for tRNA-aminoacylation, may be susceptible to oxidation resultingin an accumulation of uncharged tRNA and activation of Gcn2.

It is well established that yeast cells alter global tran-scription patterns, including genes encoding antioxidantsand other metabolic enzymes, in response to ROS (Gaschet al. 2000; Causton et al. 2001). However, given that oxi-dative stress results in a rapid and reversible inhibition ofprotein synthesis, it is unclear how changes in the geneexpression program are translated into the cellular pro-teome. Oxidative stress causes complex changes in the pat-tern of protein synthesis. Although translation of mostmRNAs is inhibited in response to oxidative stress condi-tions, certain mRNAs remain translationally active or are

Figure 9 Control of translation initiation by Gcn2. Gcn2 is activated inresponse to diverse oxidative stress conditions that may occur via anaccumulation of uncharged tRNA. Gcn2 phosphorylates eIF2, which con-verts it into a competitive inhibitor of the eIF2B guanine nucleotideexchange factor. Decreased eIF2B activity generates less eIF2 in theGTP-bound form, resulting in decreased ternary complex levels and in-hibition of translation initiation.














strongly downregulated. For example, the GCN4 mRNA isactivated via a mechanism involving short upstream openreading frames (Hinnebusch 2005). Gcn4 is a transcriptionfactor, which activates amino acid biosynthetic genes toovercome the imposed starvation which initially led to itstranslational control. Not only is Gcn4 upregulated in re-sponse to hydrogen peroxide, but it is specifically requiredfor hydroperoxide resistance (Mascarenhas et al. 2008).Transcriptional profiling studies have shown that �10% ofthe yeast genome is regulated by Gcn4 in response to aminoacid starvation, suggesting that Gcn4 acts as a master regu-lator of gene expression (Natarajan et al. 2001). However,Gcn4 is required for a more limited set of genes in responseto hydrogen peroxide stress and there is not a strong corre-lation with the amino acid starvation response at the ge-nome-wide level, suggesting that other factors moderatethe transcriptional output of Gcn4 during hydrogen perox-ide stress conditions (Mascarenhas et al. 2008).

Adaptive concentrations of hydrogen peroxide increasethe translation of several antioxidants and stress protectivemolecules. These include catalase (Ctt1) and glutathioneperoxidase (Gpx2), which can reduce H2O2 directly, thiore-doxin reductase (TRR1), which provides the reducing powerfor the thioredoxin system, and a GST (GTT2) and two GS-Xpumps (YCF1 and YBT1), which form part of the glutathioneconjugation/removal system of cells (Shenton et al. 2006). Anumber of metabolic genes are also up- or downregulated inresponse to low concentrations of H2O2, consistent with sig-nificant metabolic reconfiguration occurring during oxidativestress conditions. In contrast to adaptive concentrations ofhydrogen peroxide, high lethal concentrations of H2O2 donot appear to significantly affect the translation of antioxi-dants or other stress protective molecules (Shenton et al.2006). Prominent upregulation of genes involved in ribosomebiogenesis and rRNA processing was observed, which mayindicate a requirement to replace ribosomal proteins andrRNA that become damaged by oxidative stress. In contrast,these genes are generally transcriptionally repressed as partof the ESR (Gasch and Werner-Washburne 2002). It is un-clear why genes that are transcriptionally repressed by oxi-dants may be translationally activated but it may representa means of “fine tuning” expression levels. Additionally, a sig-nificant number of the genes that are translationally down-regulated in response high H2O2 concentrations are increasedat the transcript level. These data indicate that certain genesare increased at the transcriptional level in response to H2O2,but remain poorly translated. Increasing transcript levels inthe absence of active translation may provide a source ofmRNAs that can become rapidly translated once the stressis removed (Shenton et al. 2006).

Oxidative stress must regulate translation via additionalGcn2-independent mechanism(s), since protein synthesis isstill significantly inhibited in a gcn2 mutant (Shenton et al.2006). H2O2 causes a postinitiation inhibition of translation,increasing the average ribosomal transit time on mRNAs(Shenton et al. 2006). Regulation of mRNA expression via

translation elongation is clearly a critical component of pro-tein synthesis, but is relatively poorly understood. Oxidativestress in mammalian cells increases elongation factor 2(eEF2) phosphorylation and oxidative modification, whichis thought to contribute to translation inhibition (Patel et al.2002). Similarly, the yeast mitogen-activated protein kinase,Rck2, which phosphorylates eEF2, may affect elongatingribosomes during stress (Swaminathan et al. 2006). Expo-sure to oxidative (tert-butyl hydroperoxide) or osmoticstress was found to cause a pronounced dissociation of poly-somes in an rck2Δ mutant. Microarray analysis indicatedthat a number of weakly transcribed mRNAs associate moreavidly with polysomes during stress conditions, consistentwith a role for Rck2 in mRNA-polysome association.

While it is now becoming clearer that ribosome associa-tion and translational control of individual mRNAs can behighly regulated, much still remains to be investigatedregarding alternative fates of mRNAs during oxidative stressconditions. For example, mRNA decay dynamics are signif-icantly altered in response to oxidative stress conditions,which may contribute to downregulation of protein synthe-sis (Molina-Navarro et al. 2008). This may be explained bythe targeting of mRNAs to stress granules, which arethought to act as storage factors that sort mRNAs for deg-radation or later translation following the relief of stressconditions (Teixeira et al. 2005). Storage or degradationof mRNAs in P bodies and stress granules may play an im-portant role in regulating gene expression during oxidativestress conditions since it has been shown that the number ofP bodies is significantly increased following UV and hydro-gen peroxide stress (Teixeira et al. 2005; Mazzoni et al.2007).

Metabolic reconfiguration is a rapid, regulated responseto oxidative stress: It is well established that yeast cellsadapt to oxidative stress conditions by altering global geneexpression patterns, including transcription and translationof genes encoding antioxidants and other stress-protectivedefenses. However, it is now becoming increasingly recog-nized that post-translational changes are key regulators ofstress responses. In fact, metabolic changes are detectedwithin seconds of an oxidative stress, before slower(within minutes) changes in gene expression are measured(Chechik et al. 2008; Ralser et al. 2009). Key to these met-abolic changes appears to be the reprogramming of carbo-hydrate metabolism, which is essential to maintain theredox balance of the cell during oxidative conditions. Inparticular, dynamic rerouting of the metabolic flux from gly-colysis to the pentose phosphate pathway, with the concom-itant generation of NADPH, appears to be a conservedresponse to oxidative stress (Ralser et al. 2007).

The pentose phosphate pathway is the source of cellularreducing power in the form of NADPH. NADPH is particu-larly important during exposure to oxidants, since itprovides the reducing potential for most antioxidant andredox regulatory enzymes including the GSH/glutaredoxin




















and thioredoxin systems. Glucose 6-phosphate dehydroge-nase (G6PDH) and 6-phosphogluconate dehydrogenase(6PGDH) catalyze the first two steps of the pentosephosphate pathway and are the source of NADPH. G6PDHcatalyzes the key NADPH-production step and is known toplay a role in protection against oxidative stress (Slekar et al.1996). Additionally, G6PDH and 6PGDH enzyme activitiesare maintained in yeast cells during oxidant exposure, con-firming their role in the oxidative stress response (Izawaet al. 1998; Shenton and Grant 2003). The pentose phos-phate pathway is directly connected to glycolysis via theoxidation of glucose 6-phosphate. Hence, any growth con-dition that influences glycolytic activity can potentially alterthe flux of glucose equivalents through the pentose phos-phate pathway leading to the generation of NADPH.This has been shown experimentally, since reducing the ac-tivity of glycolytic enzymes such as triosephosphate isomer-ase (TPI) or glyceraldehyde 3-phosphate dehydrogenase(GAPDH) confers resistance against oxidative stress condi-tions (Ralser et al. 2006, 2007). Quantitative metabolomicanalysis has directly confirmed that reduced TPI or GAPDHactivity redirects glucose equivalents to the pentose phos-phate pathway, increasing the concentration of pentosephosphate pathway metabolites and shifting the NADPH/NADP+ ratio to a more reduced state (Ralser et al. 2007).

An important question is how glycolytic enzyme activityis regulated during oxidative stress conditions. Increasingevidence suggests that post-translational modification ofglycolytic enzymes is a common response to oxidative stresscausing rapid and reversible changes in enzyme activity(Biswas et al. 2006). For example, GAPDH has frequentlybeen identified as a target of oxidative modification in di-verse cellular systems, leading to the suggestion that it mayserve a regulatory role as a sensor of oxidative stress con-ditions (Chuang et al. 2005). The yeast Tdh2 and Tdh3GAPDH isoenzymes share extensive sequence homology(98% similarity, 96% identity) but appear to play distinctroles in regulating glycolytic flux in response to oxidativestress conditions (Grant et al. 1999). The Tdh3 isoenzyme,but not the Tdh2 isoenzyme, is modified by glutathionyla-tion following exposure to hydrogen peroxide. Glutathiony-lation modifies the active site cysteine residues of Tdh3 andcorrelates with an inhibition of GAPDH activity (Grant et al.1999). Several glycolytic enzymes contain oxidized cysteineresidues during normal growth conditions but only Tdh3shows an increase in oxidation in response to hydrogen per-oxide stress (Le Moan et al. 2006). Oxidation of Tdh3appears to be a very rapid response, which can be detectedwithin 1 min, before changes in gene expression occur. Irre-versible oxidative damage may also inhibit glycolytic enzymeactivity during oxidative stress conditions. Protein carbonylformation is frequently used as a measure of protein oxidativedamage and glycolytic enzymes including GAPDH, enolase,and TPI have been shown to be carbonylated in response tooxidative stress (Costa et al. 2002; Shanmuganathan et al.2004). Additionally, the synthesis of a number of glycolytic

enzymes is repressed following H2O2 exposure (Godon et al.1998). Yeast cells therefore respond to an oxidative stress byinhibiting the activity of glycolytic enzymes and by switchingoff gene expression to prevent synthesis of any new enzymes.Combining regulation at the levels of protein modificationand gene expression provides a rapid means of reversiblyinhibiting the flux through glycolysis. Additionally, irrevers-ible oxidative damage to glycolytic enzymesmay add a furtherlevel of control.

Control of the cell division cycle vs. apoptotic cell deathfollowing oxidative stress: Yeast cells can respond to ROSexposure by delaying progression through the cell divisioncycle. This may enable them to repair any macromoleculardamage without passing it on to their daughter cells(Shackelford et al. 2000). For example, hydrogen peroxidecauses a RAD9-dependent G2 cell cycle arrest by activatingthe Rad53 checkpoint (Flattery-O’Brien and Dawes 1998).The nature of the cell cycle delay is specific to the particularROS, since the superoxide anion was found to cause a G1arrest independent of Rad9 function (Nunes and Siede1996; Flattery-O’Brien and Dawes 1998). The superoxide-induced G1 arrest appears to be mediated via an inhibitionof transcription of the CLN1 and CLN2 G1 cyclins (Lee et al.1996). Similarly, LoaOOH exposure causes a G1 cell cycledelay, which is mediated by the Swi6 transcription factor(Alic et al. 2001; Fong et al. 2008). Swi6 is thought to actas a redox sensor that suppresses the expression of G1cyclins in response to oxidation of a specific cysteine residuein Swi6 (Chiu et al. 2011). Swi6 therefore provides an ex-ample of an oxidant-specific mechanism for controlling thecell cycle. However, it is not yet clear whether other oxidantcontrol mechanisms are generally unique to oxidative dam-age or whether they are more generally responses to DNAdamage initiated by ROS exposure.

ROS exposure and the resulting oxidative stress cancause a form of programmed cell death (PCD) in yeast(Madeo et al. 2002). Yeast apoptosis was triggered in re-sponse to hydrogen peroxide exposure or depletion ofGSH in gsh1 mutants leading to the hypothesis that ROSgeneration is a key component of the apoptotic pathway(Madeo et al. 1999). Subsequently, numerous stress condi-tions have been shown to promote yeast apoptosis, many ofwhich correlate with the production of ROS (Pereira et al.2008; Perrone et al. 2008). For many of these stress condi-tions, it is not clear whether ROS production is the factorthat triggers the apoptotic response or whether ROS gener-ation is a byproduct of the cell death pathway. Clear exam-ples are provided by mutants in CDC48, which normallyfunctions in retrotranslocation of ubiquitinated proteinsfrom the ER into the cytosol for degradation by the protea-some and in yeast expressing the mammalian proapoptoticprotein Bax. Apoptosis has been shown to involve an accu-mulation of ROS in both cases (Madeo et al. 1999). Impor-tantly, ROS scavengers and anaerobic growth conditionswere found to suppress PCD, confirming that at least under





















these conditions ROS generation is an important intermedi-ate in the apoptotic pathway. Yeast contains a metacaspase(Yca1), which mediates cell death in response to variousstimuli (Madeo et al. 2002; Khan et al. 2005). In the absenceof Yca1, apoptosis is suppressed and peroxide exposuredamages yeast cells, causing an accumulation of oxidizedproteins and activation of proteasome activity (Khan et al.2005). Yca1 is known to induce apoptosis in response tooxidative stress caused by hydrogen peroxide exposure,and yca1 mutants are resistant to hydrogen peroxide, con-sistent with apoptosis accounting for cell death during ROSexposure (Madeo et al. 2002; Khan et al. 2005). Yeast alsocontain a homolog of the mammalian apoptosis-inducingfactor (AIF), which regulates apoptosis in yeast (Wissinget al. 2004). Aif1 also appears to play a role in ROS-inducedapoptosis, since aif1Δ mutants are resistant to hydrogenperoxide and overexpression of AIF1 strongly stimulatesperoxide-induced apoptosis.

More work is required to decipher the mechanisms thatdictate whether yeast cells undergo a cell cycle divisiondelay vs. PCD. This decision may be related to the particularROS that yeast cells are exposed to as well as to the dosageand exposure time. Delaying the cell division cycle mayenable yeast cells to attempt to detoxify and repair ROS-mediated damage, enabling them to overcome and adaptto the stress condition. Apoptosis provides a mechanismfor the selective death of a subset of the yeast population,which can be beneficial in a unicellular organism undercertain stress conditions (Gourlay et al. 2006).

Higher concentrations of oxidants are likely to over-whelm yeast cell antioxidant defenses, causing extensivecellular damage and ultimately necrotic cell death of thepopulation.

Future directions

What is the future of research into the yeast HSR? Withgenomic and proteomic technologies providing the bird’s-eye view of the transcriptional changes and protein–proteininteractions, respectively, the volume of information obtainedin the last decade has been tremendous. The primary taskover the next decade will be to sift through these mountainsof data to validate the large-scale results and continue mak-ing connections among the various functional nodes identi-fied. As with all biological systems, a laudable but challenginggoal is to achieve predictive confidence in the outcomes ofexperimental manipulations, which requires full understand-ing of the primary, secondary, tertiary, etc., effects of a stressstimulus. Now that the response is better understood, thefocus can be applied to the products of the HSR—the stressproteins—to decipher their precise roles in stress toleranceand adaptation. The driving force behind these investigationswill likely remain the parallels between the stressed yeast celland the pathological human cell. For example, understandinghow Hsp90 regulates the activity of a number of clients re-quired for stress survival in yeast should shed light on howthe chaperone does the same thing in a tumor. Moreover,

pharmacologic and genetic manipulation in the former sce-nario will provide insight that will support therapeutic inter-vention in the latter.

As described above for the study of heat stress resistance,our understanding of the basic components and processesinvolved in supporting tolerance to oxidative stress hasexpanded greatly in recent years. However, importantquestions remain. Clear data have been provided demon-strating that expression of a variety of genes is stronglyinduced upon stress with oxidants but the precise nature ofthe signal(s) that triggers this response remains elusive.Global analyses have assembled a parts list of proteins andgenes involved in oxidative stress resistance, but recon-structing these individual constituents into their cellularcontext is a central challenge. A cell experiences oxidativestress as the sum total of the pathways considered aboveand must integrate their functions into a cogent response.This integration seems likely to be a natural applicationfor systems biology as research moves forward. It is alsoimportant that future research examines yeast cells underdifferent growth conditions. Most studies to date haveexamined cells grown under fermentative conditions usingglucose as a carbon source. These studied should be ex-tended to include respiratory growth conditions, whichwould be expected to generate intracellular ROS via themitochondrial electron transport chain. Respiratory growthconditions are also particularly relevant to mammalian cellgrowth. Additionally, many antioxidant genes are subject tocarbon catabolite repression (e.g., SOD2) and are inducedunder respiratory conditions presumably as a response toendogenously generated oxidative stress.

Oxidative stress is often thought of as an externallyimposed challenge but every eukaryotic cell contains oxida-tive environments such as the endoplasmic reticulum andmitochondria within its cytoplasm. Details underlying thecommunication between and coordination of the oxidativecontents of these organelles and the reductive nature of therest of the cell are obscure. Key questions remain regardinghow cells maintain compartment-specific redox regulationwhile protecting against oxidative damage to other compo-nents of the cell. The discovery of apoptotic machinery in S.cerevisiae provides the ability to employ the facile geneticanalysis of the organism to explore this fundamental eukary-otic process. The yeast system should provide a model toidentify the functional links between ROS and fungal apo-ptosis. Given the past record of successful investigations in S.cerevisiae, we are confident that future studies of oxidativestress tolerance will prove fruitful in expanding the under-standing of these and other areas of eukaryotic cell biology.

Literature Cited

Adachi, Y., and M. Yanagida, 1989 Higher order chromosomestructure is affected by cold-sensitive mutations in a Schizosac-charomyces pombe gene crm1+ which encodes a 115-kDa pro-tein preferentially localized in the nucleus and its periphery.J. Cell Biol. 108: 1195–1207.










Ahn, S. G., and D. J. Thiele, 2003 Redox regulation of mammalianheat shock factor 1 is essential for Hsp gene activation and pro-tection from stress. Genes Dev. 17: 516–528.

Ahn, S. G., P. C. Liu, K. Klyachko, R. I. Morimoto, and D. J. Thiele,2001 The loop domain of heat shock transcription factor 1dictates DNA-binding specificity and responses to heat stress.Genes Dev. 15: 2134–2145.

Akerfelt, M., R. I. Morimoto, and L. Sistonen, 2010 Heat shockfactors: integrators of cell stress, development and lifespan. Nat.Rev. Mol. Cell Biol. 11: 545–555.

Albanese, V., A. Y. Yam, J. Baughman, C. Parnot, and J. Frydman,2006 Systems analyses reveal two chaperone networks withdistinct functions in eukaryotic cells. Cell 124: 75–88.

Alejandro-Osorio, A. L., D. J. Huebert, D. T. Porcaro, M. E. Sonntag,S. Nillasithanukroh et al., 2009 The histone deacetylaseRpd3p is required for transient changes in genomic expressionin response to stress. Genome Biol. 10: R57.

Alic, A., V. J. Higgins, and I. W. Dawes, 2001 Identification ofa Saccharomyces cerevisiae gene that Is required for G1 arrestin response to the lipid oxidation product linoleic acid hydro-peroxide. Mol. Biol. Cell 12: 1802–1810.

Avery, A. M., and S. V. Avery, 2001 Saccharomyces cerevisiae ex-presses three phospholipid hydroperoxide glutathione peroxi-dases. J. Biol. Chem. 276: 33730–33735.

Avery, A. M., S. A. Willetts, and S. V. Avery, 2004 Genetic dissec-tion of the phospholipid hydroperoxidase activity of yeast gpx3reveals its functional importance. J. Biol. Chem. 279: 46652–46658.

Azevedo, D., L. Nascimento, J. Labarre, M. B. Toledano, and C.Rodrigues-Pousada, 2007 The S. cerevisiae Yap1 and Yap2transcription factors share a common cadmium-sensing domain.FEBS Lett. 581: 187–195.

Barreto, L., A. Garcera, K. Jansson, P. Sunnerhagen, and E. Herrero,2006 A peroxisomal glutathione transferase of Saccharomycescerevisiae is functionally related to sulfur amino acid metabo-lism. Eukaryot. Cell 5: 1748–1759.

Baumann, F., I. Milisav, W. Neupert, and J. M. Herrmann,2000 Ecm10, a novel hsp70 homolog in the mitochondrial matrixof the yeast Saccharomyces cerevisiae. FEBS Lett. 487: 307–312.

Baxter, B. K., P. James, T. Evans, and E. A. Craig, 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum. Mol. Cell. Biol. 16: 6444–6456.

Berry, D. B., and A. P. Gasch, 2008 Stress-activated genomic ex-pression changes serve a preparative role for impending stress inyeast. Mol. Biol. Cell 19: 4580–4587.

Biswas, S., A. S. Chida, and I. Rahman, 2006 Redox modificationsof protein-thiols: emerging roles in cell signaling. Biochem.Pharmacol. 71: 551–564.

Biteau, B., J. Labarre, and M. B. Toledano, 2003 ATP-dependentreduction of cysteine-sulphinic acid by S. cerevisiae sulphire-doxin. Nature 425: 980–984.

Bohen, S. P., 1998 Genetic and biochemical analysis of p23 andansamycin antibiotics in the function of Hsp90-dependent sig-naling proteins. Mol. Cell. Biol. 18: 3330–3339.

Boisnard, S., G. Lagniel, C. Garmendia-Torres, M. Molin, E. Boy-Marcotte et al., 2009 H2O2 activates the nuclear localizationof Msn2 and Maf1 through thioredoxins in Saccharomyces cer-evisiae. Eukaryot. Cell 8: 1429–1438.

Borkovich, K. A., F. W. Farrelly, D. B. Finkelstein, J. Taulien, and S.Lindquist, 1989 hsp82 is an essential protein that is requiredin higher concentrations for growth of cells at higher temper-atures. Mol. Cell. Biol. 9: 3919–3930.

Bose, S., J. A. Dutko, and R. S. Zitomer, 2005 Genetic factors thatregulate the attenuation of the general stress response of yeast.Genetics 169: 1215–1226.

Bossier, P., L. Fernandes, D. Rocha, and C. Rodrigues-Pousada,1993 Overexpression of YAP2, coding for a new yAP protein,

and YAP1 in S. cerevisiae alleviates growth inhibition caused by1,10-phenanthroline. J. Biol. Chem. 268: 23640–23645.

Brennan, R. J., and R. H. Schiestl, 1996 Cadmium is an inducer ofoxidative stress in yeast. Mutat. Res. 356: 171–178.

Brodsky, J. L., E. D. Werner, M. E. Dubas, J. L. Goeckeler, K. B. Kruseet al., 1999 The requirement for molecular chaperones duringendoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct. J. Biol. Chem. 274: 3453–3460.

Brown, J. L., S. North, and H. Bussey, 1993 SKN7, a yeast multi-copy suppressor of a mutation affecting cell wall beta-glucanassembly, encodes a product with domains homologous to pro-karyotic two-component regulators and to heat shock transcrip-tion factors. J. Bacteriol. 175: 6908–6915.

Camier, S., E. Ma, C. Leroy, A. Pruvost, M. Toledano et al.,2007 Visualization of ribonucleotide reductase catalytic oxida-tion establishes thioredoxins as its major reductants in yeast.Free Radic. Biol. Med. 42: 1008–1016.

Castells-Roca, L., J. Garcia-Martinez, J. Moreno, E. Herrero, G. Belliet al., 2011 Heat shock response in yeast involves changes inboth transcription rates and mRNA stabilities. PLoS ONE 6:e17272.

Causton, H. C., B. Ren, S. S. Koh, C. T. Harbison, E. Kanin et al.,2001 Remodeling of yeast genome expression in response toenvironmental changes. Mol. Biol. Cell 12: 323–337.

Cha, M. K., Y. S. Choi, S. K. Hong, W. C. Kim, K. T. No et al.,2003 Nuclear thiol peroxidase as a functional alkyl-hydroper-oxide reductase necessary for stationary phase growth of Sac-charomyces cerevisiae. J. Biol. Chem. 278: 24636–24643.

Chae, H. Z., S. J. Chung, and S. G. Rhee, 1994 Thioredoxin-dependent peroxide reductase from yeast. J. Biol. Chem. 269:27670–27678.

Chang, H. C., D. F. Nathan, and S. Lindquist, 1997 In vivo analysisof the Hsp90 cochaperone Sti1 (p60). Mol. Cell. Biol. 17: 318–325.

Charizanis, C., H. Juhnke, B. Krems, and K. D. Entian, 1999a Themitochondrial cytochrome c peroxidase Ccp1 of Saccharomycescerevisiae is involved in conveying an oxidative stress signal tothe transcription factor Pos9 (Skn7). Mol. Gen. Genet. 262:437–447.

Charizanis, C., H. Juhnke, B. Krems, and K. D. Entian, 1999b Theoxidative stress response mediated via Pos9/Skn7 is negativelyregulated by the Ras/PKA pathway in Saccharomyces cerevisiae.Mol. Gen. Genet. 261: 740–752.

Chechik, G., E. Oh, O. Rando, J. Weissman, A. Regev et al.,2008 Activity motifs reveal principles of timing in transcrip-tional control of the yeast metabolic network. Nat. Biotechnol.26: 1251–1259.

Chernoff, Y. O., S. L. Lindquist, B. Ono, S. G. Inge-Vechtomov, andS. W. Liebman, 1995 Role of the chaperone protein Hsp104 inpropagation of the yeast prion-like factor [psi+]. Science 268:880–884.

Chi, Y., M. J. Huddleston, X. Zhang, R. A. Young, R. S. Annan et al.,2001 Negative regulation of Gcn4 and Msn2 transcription factorsby Srb10 cyclin-dependent kinase. Genes Dev. 15: 1078–1092.

Chiu, J., C. M. Tactacan, S. X. Tan, R. C. Lin, M. A. Wouters et al.,2011 Cell cycle sensing of oxidative stress in Saccharomycescerevisiae by oxidation of a specific cysteine residue in the tran-scription factor Swi6p. J. Biol. Chem. 286: 5204–5214.

Choi, J. H., W. Lou, and A. Vancura, 1998 A novel membrane-bound glutathione S-transferase functions in the stationaryphase of the yeast Saccharomyces cerevisiae. J. Biol. Chem.273: 29915–29922.

Chuang, D. M., C. Hough, and V. V. Senatorov,2005 Glyceraldehyde-3-phosphate dehydrogenase, apoptosis,and neurodegenerative diseases. Annu. Rev. Pharmacol. Toxicol.45: 269–290.


Chughtai, Z. S., R. Rassadi, N. Matusiewicz, and U. Stochaj,2001 Starvation promotes nuclear accumulation of the hsp70Ssa4p in yeast cells. J. Biol. Chem. 276: 20261–20266.

Cohen, T. J., K. Lee, L. H. Rutkowski, and R. Strich, 2003 Ask10pmediates the oxidative stress-induced destruction of the Saccha-romyces cerevisiae C-type cyclin Ume3p/Srb11p. Eukaryot. Cell2: 962–970.

Coleman, S. T., E. A. Epping, S. M. Steggerda, and W. S. Moye-Rowley, 1999 Yap1p activates gene transcription in an oxi-dant-specific fashion. Mol. Cell. Biol. 19: 8302–8313.

Collinson, E. J., and C. M. Grant, 2003 Role of yeast glutaredoxinsas glutathione S-transferases. J. Biol. Chem. 278: 22492–22497.

Collinson, E. J., G. Wheeler, E. O. Garrido, A. M. Avery, S. V. Averyet al., 2002 The yeast glutaredoxins are active as glutathioneperoxidases. J. Biol. Chem. 277: 16712–16717.

Collinson, L. P., and I. W. Dawes, 1992 Inducibilty of the responseof yeast cells to peroxide stress. J. Gen. Micro. 138: 329–335.

Cooper, K. F., and R. Strich, 2002 Saccharomyces cerevisiaeC-type cyclin Ume3p/Srb11p is required for efficient inductionand execution of meiotic development. Eukaryot. Cell 1: 66–74.

Cooper, K. F., M. J. Mallory, and R. Strich, 1999 Oxidative stress-induced destruction of the yeast C-type cyclin Ume3p requiresphosphatidylinositol-specific phospholipase C and the 26S pro-teasome. Mol. Cell. Biol. 19: 3338–3348.

Costa, V. M. V., M. A. Amorim, A. Quintanilha, and P. Moradas-Ferreira, 2002 Hydrogen peroxide-induced carbonylation ofkey metabolic enzymes in Saccharomyces cerevisiae: the in-volvement of the oxidative stress response regulators Yap1and Skn7. Free Radic. Biol. Med. 33: 1507–1515.

Cotgreave, I. A., and R. G. Gerdes, 1998 Recent trends in gluta-thione biochemistry-glutathione-protein interactions: A molecu-lar link between oxidative stress and cell proliferation?Biochem. Biophys. Res. Commun. 242: 1–9.

Cowen, L. E., 2009 Hsp90 orchestrates stress response signalinggoverning fungal drug resistance. PLoS Pathog. 5: e1000471.

Cowen, L. E., S. D. Singh, J. R. Kohler, C. Collins, A. K. Zaas et al.,2009 Harnessing Hsp90 function as a powerful, broadly effec-tive therapeutic strategy for fungal infectious disease. Proc. Natl.Acad. Sci. USA 106: 2818–2823.

Cox, J. S., and P. Walter, 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response. Cell 87: 391–404.

Craig, E. A., and C. A. Gross, 1991 Is hsp70 the cellular thermom-eter? Trends Biochem. Sci. 16: 135–140.

Craig, E. A., J. Kramer, J. Shilling, M. Werner-Washburne, S.Holmes et al., 1989 SSC1, an essential member of the yeastHSP70 multigene family, encodes a mitochondrial protein. Mol.Cell. Biol. 9: 3000–3008.

Craven, R. A., M. Egerton, and C. J. Stirling, 1996 A novel Hsp70of the yeast ER lumen is required for the efficient translocationof a number of protein precursors. EMBO J. 15: 2640–2650.

Crowe, J. H., 2007 Trehalose as a “chemical chaperone”: fact andfantasy. Adv. Exp. Med. Biol. 594: 143–158.

Culotta, V. C., 2000 Superoxide dismutase, oxidative stress, andcell metabolism. Curr. Top. Cell. Regul. 36: 117–132.

Culotta, V. C., M. Yang, and T. V. O’Halloran, 2006 Activation ofsuperoxide dismutases: putting the metal to the pedal. Biochim.Biophys. Acta 1763: 747–758.

D’Autréaux, B., and M. B. Toledano, 2007 ROS as signalling mol-ecules: mechanisms that generate specificity in ROS homeosta-sis. Nat. Rev. Mol. Cell Biol. 8: 813–824.

Davidson, J. F., and R. H. Schiestl, 2001 Mitochondrial respira-tory electron carriers are involved in oxidative stress during heatstress in Saccharomyces cerevisiae. Mol. Cell. Biol. 21: 8483–8489.

Davidson, J. F., B. Whyte, P. H. Bissinger, and R. H. Schiestl,1996 Oxidative stress is involved in heat-induced cell death

in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 93:5116–5121.

De Freitas, J., H. Wintz, J. H. Kim, H. Poynton, T. Fox et al.,2003 Yeast, a model organism for iron and copper metabolismstudies. Biometals 16: 185–197.

De Wever, V., W. Reiter, A. Ballarini, G. Ammerer, and C. Brocard,2005 A dual role for PP1 in shaping the Msn2-dependent tran-scriptional response to glucose starvation. EMBO J. 24: 4115–4123.

Dean, R. T., S. Fu, R. Stocker, and M. J. Davies, 1997 Bio-chemistry and pathology of radical-mediated protein oxidation.Biochem. J. 324: 1–18.

Delaunay, A., A. D. Isnard, and M. B. Toledano, 2000 H2O2 sens-ing through oxidation of the Yap1 transcription factor. EMBO J.19: 5157–5166.

Delaunay, A., D. Pflieger, M. B. Barrault, J. Vinh, and M. B. Tole-dano, 2002 A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell 111: 471–481.

Dey, B., J. J. Lightbody, and F. Boschelli, 1996 CDC37 is requiredfor p60v-src activity in yeast. Mol. Biol. Cell 7: 1405–1417.

Dittmar, K. D., D. R. Demady, L. F. Stancato, P. Krishna, and W. B.Pratt, 1997 Folding of the glucocorticoid receptor by the heatshock protein (hsp) 90-based chaperone machinery. The role ofp23 is to stabilize receptor.hsp90 heterocomplexes formed byhsp90.p60.hsp70. J. Biol. Chem. 272: 21213–21220.

Dolinski, K. J., M. E. Cardenas, and J. Heitman, 1998 CNS1 enc-odes an essential p60/Sti1 homolog in Saccharomyces cerevi-siae that suppresses cyclophilin 40 mutations and interacts withHsp90. Mol. Cell. Biol. 18: 7344–7352.

Dormer, U. H., J. Westwater, D. W. Stephen, and D. J. Jamieson,2002 Oxidant regulation of the Saccharomyces cerevisiaeGSH1 gene. Biochim. Biophys. Acta 1576: 23–29.

Doyle, S. M., and S. Wickner, 2009 Hsp104 and ClpB: proteindisaggregating machines. Trends Biochem. Sci. 34: 40–48.

Dragovic, Z., S. A. Broadley, Y. Shomura, A. Bracher, and F. U.Hartl, 2006 Molecular chaperones of the Hsp110 family actas nucleotide exchange factors of Hsp70s. EMBO J. 25: 2519–2528.

Duina, A. A., H. C. Chang, J. A. Marsh, S. Lindquist, and R. F. Gaber,1996 A cyclophilin function in Hsp90-dependent signal trans-duction. Science 274: 1713–1715.

Duina, A. A., H. M. Kalton, and R. F. Gaber, 1998 Requirement forHsp90 and a CyP-40-type cyclophilin in negative regulation ofthe heat shock response. J. Biol. Chem. 273: 18974–18978.

Durchschlag, E., W. Reiter, G. Ammerer, and C. Schuller,2004 Nuclear localization destabilizes the stress-regulatedtranscription factor Msn2. J. Biol. Chem. 279: 55425–55432.

Dutkiewicz, R., B. Schilke, H. Knieszner, W. Walter, E. A. Craiget al., 2003 Ssq1, a mitochondrial Hsp70 involved in iron-sulfur (Fe/S) center biogenesis. Similarities to and differencesfrom its bacterial counterpart. J. Biol. Chem. 278: 29719–29727.

Easton, D. P., Y. Kaneko, and J. R. Subjeck, 2000 The hsp110 andGrp1 70 stress proteins: newly recognized relatives of theHsp70s. Cell Stress Chaperones 5: 276–290.

Eckers, E., M. Bien, V. Stroobant, J. M. Herrmann, and M. Deponte,2009 Biochemical characterization of dithiol glutaredoxin 8from Saccharomyces cerevisiae: the catalytic redox mechanismredux. Biochemistry 48: 1410–1423.

Elmlund, H., V. Baraznenok, M. Lindahl, C. O. Samuelsen, P. J.Koeck et al., 2006 The cyclin-dependent kinase 8 module ste-rically blocks Mediator interactions with RNA polymerase II.Proc. Natl. Acad. Sci. USA 103: 15788–15793.

Enjalbert, B., A. Nantel, and M. Whiteway, 2003 Stress-inducedgene expression in Candida albicans: absence of a general stressresponse. Mol. Biol. Cell 14: 1460–1467.

Erkine, A. M., S. F. Magrogan, E. A. Sekinger, and D. S. Gross,1999 Cooperative binding of heat shock factor to the yeast


HSP82 promoter in vivo and in vitro. Mol. Cell. Biol. 19: 1627–1639.

Evans, M. V., H. E. Turton, C. M. Grant, and I. W. Dawes,1997 Toxicity of linoleic acid hydroperoxide to Saccharomycescerevisiae: Involvement of a respiration-related process for max-imal sensitivity and adaptive response. J. Bacteriol. 180: 483–490.

Fang, Y., A. E. Fliss, J. Rao, and A. J. Caplan, 1998 SBA1 encodesa yeast hsp90 cochaperone that is homologous to vertebrate p23proteins. Mol. Cell. Biol. 18: 3727–3734.

Feldheim, D., J. Rothblatt, and R. Schekman, 1992 Topology andfunctional domains of Sec63p, an endoplasmic reticulum mem-brane protein required for secretory protein translocation. Mol.Cell. Biol. 12: 3288–3296.

Fernandes, L., C. Rorigues-Pousada, and K. Struhl, 1997 Yap,a novel family of eight bZIP proteins in Saccharomyces cerevisiaewith distinct biological functions. Mol. Cell. Biol. 17: 6982–6993.

Flattery-O’Brien, J. A., and I. W. Dawes, 1998 Hydrogen peroxidecauses RAD9-dependent cell cycle arrest in G2 in Saccharomycescerevisiae whereas menadione causes G1 arrest independent ofRAD9 function. J. Biol. Chem. 273: 8564–8571.

Flattery-O’Brien, J., L. P. Collinson, and I. W. Dawes,1993 Saccharomyces cerevisiae has an inducible response tomenadione which differs to that to hydrogen peroxide. J. Gen.Microbiol. 139: 501–507.

Fong, C. S., M. D. Temple, N. Alic, J. Chiu, M. Durchdewald et al.,2008 Oxidant-induced cell-cycle delay in Saccharomyces cer-evisiae: the involvement of the SWI6 transcription factor. FEM.Yeast Res. 8: 386–399.

Frydman, J., 2001 Folding of newly translated proteins in vivo: therole of molecular chaperones. Annu. Rev. Biochem. 70: 603–647.

Gall, W. E., M. A. Higginbotham, C. Chen, M. F. Ingram, D. M. Cyret al., 2000 The auxilin-like phosphoprotein Swa2p is requiredfor clathrin function in yeast. Curr. Biol. 10: 1349–1358.

Gan, Z., 1991 Yeast thioredoxin genes. J. Biol. Chem. 266: 1692–1696.

Garcera, A., L. Barreto, L. Piedrafita, J. Tamarit, and E. Herrero,2006 Saccharomyces cerevisiae cells have three Omega classglutathione S-transferases acting as 1-Cys thiol transferases. Bi-ochem. J. 398: 187–196.

Garreau, H., R. N. Hasan, G. Renault, F. Estruch, E. Boy-Marcotteet al., 2000 Hyperphosphorylation of Msn2p and Msn4p inresponse to heat shock and the diauxic shift is inhibited by cAMPin Saccharomyces cerevisiae. Microbiology 146(Pt 9): 2113–2120.

Garrett, S., and J. Broach, 1989 Loss of Ras activity in Saccharo-myces cerevisiae is suppressed by disruptions of a new kinasegene, YAKI, whose product may act downstream of the cAMP-dependent protein kinase. Genes Dev. 3: 1336–1348.

Garrido, E. O., and C. M. Grant, 2002 Role of thioredoxins in theresponse of Saccharomyces cerevisiae to oxidative stress inducedby hydroperoxides. Mol. Microbiol. 43: 993–1003.

Gasch, A. P., and M. Werner-Washburne, 2002 The genomics ofyeast responses to environmental stress and starvation. Funct.Integr. Genomics 2: 181–192.

Gasch, A. P., P. T. Spellman, C. M. Kao, O. Carmel-Harel, M. B.Eisen et al., 2000 Genomic expression programs in the re-sponse of yeast cells to environmental changes. Mol. Biol. Cell11: 4241–4257.

Gautschi, M., H. Lilie, U. Funfschilling, A. Mun, S. Ross et al.,2001 RAC, a stable ribosome-associated complex in yeastformed by the DnaK-DnaJ homologs Ssz1p and zuotin. Proc.Natl. Acad. Sci. USA 98: 3762–3767.

Gautschi, M., A. Mun, S. Ross, and S. Rospert, 2002 A functionalchaperone triad on the yeast ribosome. Proc. Natl. Acad. Sci.USA 99: 4209–4214.

Godon, C., G. Lagniel, J. Lee, J.-M. Buhler, S. Kieffer et al.,1998 The H2O2 stimulon in Saccharomyces cerevisiae. J. Biol.Chem. 273: 22480–22489.

Gong, Y., Y. Kakihara, N. Krogan, J. Greenblatt, A. Emili et al.,2009 An atlas of chaperone-protein interactions in Saccharo-myces cerevisiae: implications to protein folding pathways inthe cell. Mol. Syst. Biol. 5: 275.

Gorner, W., E. Durchschlag, M. T. Martinez-Pastor, F. Estruch, G.Ammerer et al., 1998 Nuclear localization of the C2H2 zincfinger protein Msn2p is regulated by stress and protein kinaseA activity. Genes Dev. 12: 586–597.

Gorner, W., E. Durchschlag, J. Wolf, E. L. Brown, G. Ammerer et al.,2002 Acute glucose starvation activates the nuclear localiza-tion signal of a stress-specific yeast transcription factor. EMBO J.21: 135–144.

Gourlay, C. W., W. Du, and K. R. Ayscough, 2006 Apoptosis inyeast–mechanisms and benefits to a unicellular organism. Mol.Microbiol. 62: 1515–1521.

Granneman, S., M. R. Nandineni, and S. J. Baserga, 2005 Theputative NTPase Fap7 mediates cytoplasmic 20S pre-rRNA pro-cessing through a direct interaction with Rps14. Mol. Cell. Biol.25: 10352–10364.

Grant, C. M., L. P. Collinson, J.-H. Roe, and I. W. Dawes,1996a Yeast glutathione reductase is required for protectionagainst oxidative stress and is a target gene for yAP-1 transcrip-tional regulation. Mol. Microbiol. 21: 171–179.

Grant, C. M., F. H. MacIver, and I. W. Dawes, 1996b Glutathioneis an essential metabolite required for resistance to oxidativestress in the yeast Saccharomyces cerevisiae. Curr. Genet. 29:511–515.

Grant, C. M., F. M. MacIver, and I. W. Dawes, 1996c Stationaryphase induction of GLR1 expression is mediated by the yAP-1transcriptional protein in Saccharomyces cerevisiae. Mol. Micro-biol. 22: 739–746.

Grant, C. M., F. H. MacIver, and I. W. Dawes, 1997 Glutathionesynthetase is dispensable for growth under both normal andoxidative stress conditions in the yeast Saccharomyces cerevisiaedue to an accumulation of the dipeptide gamma-glutamylcys-teine. Mol. Biol. Cell 8: 1699–1707.

Grant, C. M., G. Perrone, and I. W. Dawes, 1998 Glutathione andcatalase provide overlapping defenses for protection against hy-drogen peroxide in the yeast Saccharomyces cerevisiae. Biochem.Biophys. Res. Commun. 253: 893–898.

Grant, C. M., K. A. Quinn, and I. W. Dawes, 1999 Differentialprotein S-thiolation of Glyceraldhyde 3-phosphate dehydroge-nase isoenzymes influences sensitivity to oxidative stress. Mol.Cell. Biol. 19: 2650–2656.

Grant, C. M., S. Luikenhuis, A. Beckhouse, M. Soderbergh, and I. W.Dawes, 2000 Differential regulation of glutaredoxin gene ex-pression in response to stress conditions in the yeast Saccharo-myces cerevisiae. Biochim. Biophys. Acta 1490: 33–42.

Greetham, D., and C. M. Grant, 2009 Antioxidant activity of theyeast mitochondrial 1-Cys peroxiredoxin is dependent on thio-redoxin reductase and glutathione in vivo. Mol. Cell. Biol. 29:3229–3240.

Greetham, D., J. Vickerstaff, D. Shenton, G. G. Perrone, I. W. Daweset al., 2010 Thioredoxins function as deglutathionylase en-zymes in the yeast Saccharomyces cerevisiae. BMC Biochem.11: 3.

Griffioen, G., P. Branduardi, A. Ballarini, P. Anghileri, J. Norbecket al., 2001 Nucleocytoplasmic distribution of budding yeastprotein kinase A regulatory subunit Bcy1 requires Zds1 and isregulated by Yak1-dependent phosphorylation of its targetingdomain. Mol. Cell. Biol. 21: 511–523.

Gross, D. S., K. E. English, K. W. Collins, and S. W. Lee,1990 Genomic footprinting of the yeast HSP82 promoterreveals marked distortion of the DNA helix and constitutive


occupancy of heat shock and TATA elements. J. Mol. Biol. 216:611–631.

Gross, D. S., C. C. Adams, S. Lee, and B. Stentz, 1993 A criticalrole for heat shock transcription factor in establishing a nucleo-some-free region over the TATA-initiation site of the yeastHSP82 heat shock gene. EMBO J. 12: 3931–3945.

Grousl, T., P. Ivanov, I. Frydlova, P. Vasicova, F. Janda et al.,2009 Robust heat shock induces eIF2alpha-phosphorylation-independent assembly of stress granules containing eIF3 and40S ribosomal subunits in budding yeast, Saccharomyces cere-visiae. J. Cell Sci. 122: 2078–2088.

Guidot, D. M., J. M. McCord, R. M. Wright, and J. E. Repine,1993 Absence of electron transport (Rhoo) state) restoresgrowth of a manganese-superoxide dismutase-deficient Saccha-romyces cerevisiae in hyperoxia. J. Biol. Chem. 268: 26699–26703.

Gulshan, K., S. A. Rovinsky, and W. S. Moye-Rowley, 2004 YBP1and its homologue YBP2/YBH1 influence oxidative-stress toler-ance by nonidentical mechanisms in Saccharomyces cerevisiae.Eukaryot. Cell 3: 318–330.

Gulshan, K., S. A. Rovinsky, S. T. Coleman, and W. S. Moye-Rowley,2005 Oxidant-specific folding of Yap1p regulates both tran-scriptional activation and nuclear localization. J. Biol. Chem.280: 40524–40533.

Hahn, J. S., and D. J. Thiele, 2004 Activation of the Saccharomy-ces cerevisiae heat shock transcription factor under glucose star-vation conditions by Snf1 protein kinase. J. Biol. Chem. 279:5169–5176.

Hahn, J. S., Z. Hu, D. J. Thiele, and V. R. Iyer, 2004 Genome-wideanalysis of the biology of stress responses through heat shocktranscription factor. Mol. Cell. Biol. 24: 5249–5256.

Halliwell, B., 2006 Reactive species and antioxidants. Redox bi-ology is a fundamental theme of aerobic life. Plant Physiol. 141:312–322.

Hamilton, T. G., and G. C. Flynn, 1996 Cer1p, a novel Hsp70-related protein required for posttranslational endoplasmic retic-ulum translocation in yeast. J. Biol. Chem. 271: 30610–30613.

Harding, H. P., I. Novoa, Y. Zhang, H. Zeng, R. Wek et al.,2000 Regulated translation initiation controls stress-inducedgene expression in mammalian cells. Mol. Cell 6: 1099–1108.

Hardings, C. O., P. Williams, E. Wagner, D. S. Chang, K. Wild et al.,1997 Mice with genetic gamma-glutamyl transpeptidase defi-ciency exhibit glutathionuria, severe growth failure, reduced lifespans and infertility. J. Biol. Chem. 272: 12560–12567.

Harris, N., M. MacLean, K. Hatzianthis, B. Panaretou, and P. W.Piper, 2001 Increasing Saccharomyces cerevisiae stress resis-tance, through the overactivation of the heat shock responseresulting from defects in the Hsp90 chaperone, does not extendreplicative life span but can be associated with slower chrono-logical ageing of nondividing cells. Mol. Genet. Genomics 265:258–263.

Harshman, K. D., W. S. Moye-Rowley, and C. S. Parker,1988 Transcriptional activation by the SV40 AP-1 recognitionelement in yeast is mediated by a factor similar to AP-1 that isdistinct from GCN4. Cell 53: 321–330.

Hashikawa, N., Y. Mizukami, H. Imazu, and H. Sakurai,2006 Mutated yeast heat shock transcription factor activatestranscription independently of hyperphosphorylation. J. Biol.Chem. 281: 3936–3942.

Haslbeck, M., S. Walke, T. Stromer, M. Ehrnsperger, H. E. Whiteet al., 1999 Hsp26: a temperature-regulated chaperone. EMBOJ. 18: 6744–6751.

Haslbeck, M., N. Braun, T. Stromer, B. Richter, N. Model et al.,2004 Hsp42 is the general small heat shock protein in thecytosol of Saccharomyces cerevisiae. EMBO J. 23: 638–649.

Haslbeck, M., A. Miess, T. Stromer, S. Walter, and J. Buchner,2005 Disassembling protein aggregates in the yeast cytosol.

The cooperation of Hsp26 with Ssa1 and Hsp104. J. Biol. Chem.280: 23861–23868.

He, X. J., K. E. Mulford, and J. S. Fassler, 2009 Oxidative stressfunction of the Saccharomyces cerevisiae Skn7 receiver domain.Eukaryot. Cell 8: 768–778.

Hiltunen, J. K., A. M. Mursula, H. Rottensteiner, R. K. Wierenga, A.J. Kastaniotis et al., 2003 The biochemistry of peroxisomalbeta-oxidation in the yeast Saccharomyces cerevisiae. FEMS Mi-crobiol. Rev. 27: 35–64.

Hinnebusch, A. G., 2005 Translational regulation of gcn4 and thegeneral amino Acid control of yeast. Annu. Rev. Microbiol. 59:407–450.

Hirata, D., K. Yano, and T. Miyakawa, 1994 Stress-induced tran-scriptional activation mediated by YAP1 and YAP2 genes thatencode the Jun family of transcriptional activators in Saccharo-myces cerevisiae. Mol. Gen. Genet. 242: 250–256.

Ho, A. K., G. A. Raczniak, E. B. Ives, and S. R. Wente, 1998 Theintegral membrane protein snl1p is genetically linked to yeastnuclear pore complex function. Mol. Biol. Cell 9: 355–373.

Holmgren, A., 1989 Thioredoxin and glutaredoxin systems. J.Biol. Chem. 264: 13963–13966.

Holstege, F. C. P., E. G. Jennings, J. J. Wyrick, T. I. Lee, C. J.Hengartner et al., 1998 Dissecting the regulatory circuitry ofa eukaryotic genome. Cell 95: 717–728.

Huh, W. K., B. H. Lee, S. T. Kim, Y. R. Kim, G. E. Rhie et al.,1998 D-Erythroascorbic acid is an important antioxidant mol-ecule in Saccharomyces cerevisiae. Mol. Microbiol. 30: 895–903.

Hundley, H., H. Eisenman, W. Walter, T. Evans, Y. Hotokezakaet al., 2002 The in vivo function of the ribosome-associatedHsp70, Ssz1, does not require its putative peptide-binding do-main. Proc. Natl. Acad. Sci. USA 99: 4203–4208.

Hussain, M., and J. Lenard, 1991 Characterization of PDR4,a Saccharomyces cerevisiae gene that confers pleiotropic drugresistance in high-copy number. Gene 101: 149–152.

Immenschuh, S., and E. Baumgart-Vogt, 2005 Peroxiredoxins, ox-idative stress, and cell proliferation. Antioxid. Redox Signal. 7:768–777.

Inoue, Y., T. Matsuda, K. Sugiyama, S. Iszawa, and A. Kimura,1999a Genetic analysis of glutathione peroxidase in oxidativestress response of Saccharomyces cerevisiae. J. Biol. Chem. 274:27002–27009.

Inoue, Y., T. Matsuda, K.-I. Sugiyama, S. Izawa, and A. Kimura,1999b Genetic analysis of glutathione peroxidase in oxidativestress response of Saccharomyces cerevisiae. J. Biol. Chem. 274:27002–27009.

Isoyama, T., A. Murayama, A. Nomoto, and S. Kuge, 2001 Nu-clear import of yeast AP-1-like transcription factor Yap1p is me-diated by transport receptor Pse1p and this import step is notaffected by oxidative stress. J. Biol. Chem. 276: 21863–21869.

Iwai, K., A. Naganuma, and S. Kuge, 2010 Peroxiredoxin Ahp1acts as a receptor for alkylhydroperoxides to induce disulfidebond formation in the Cad1 transcription factor. J. Biol. Chem.285: 10597–10604.

Izawa, S., Y. Inoue, and A. Kimura, 1996 Importance of catalasein the adaptive response to hydrogen peroxide: analysis of aca-talasaemic Saccharomyces cerevisae. Biochem. J. 320: 61–67.

Izawa, S., K. Maeda, T. Miki, J. Mano, Y. Inoue et al., 1998 Im-portance of glucose-6-phosphate dehydrogenase in the adaptiveresponse to hydrogen peroxide in Saccharomyces cerevisiae. Bio-chem. J. 330: 811–817.

Izawa, S., K. Maeda, K.-I. Sugiyama, J. Mano, Y. Inoue et al.,1999 Thioredoxin derficiency causes the constitutive activa-tion of Yap1, an AP-1-like transcription factor in Saccharomycescerevisiae. J. Biol. Chem. 274: 28459–28465.

Izawa, S., N. Kuroki, and Y. Inoue, 2004 Nuclear thioredoxinperoxidase Dot5 in Saccharomyces cerevisiae: roles in oxidative


stress response and disruption of telomeric silencing. Appl. Mi-crobiol. Biotechnol. 64: 120–124.

Izquierdo, A., C. Casas, U. Mühlenhoff, C. H. Lillig, and E. Herrero,2008 Yeast Grx6 and Grx7 are monothiol glutaredoxins asso-ciated with the early secretory pathway. Eukaryot. Cell 7: 1415–1426.

Jacquet, M., G. Renault, S. Lallet, J. De Mey, and A. Goldbeter,2003 Oscillatory nucleocytoplasmic shuttling of the generalstress response transcriptional activators Msn2 and Msn4 inSaccharomyces cerevisiae. J. Cell Biol. 161: 497–505.

Jain, A., J. Martensson, E. Einar, P. A. M. Auld, and A. Meister,1991 Glutathione deficiency leads to mitochondrial damagein brain. Proc. Natl. Acad. Sci. USA 88: 1913–1917.

Jamieson, D. J., 1992 Saccharomyces cerevisiae has distinct adap-tive responses to both hydrogen peroxide and menadione. J.Bacteriol. 174: 6678–6681.

Jang, H. H., K. O. Lee, Y. H. Chi, B. G. Jung, S. K. Park et al.,2004 Two enzymes in one; two yeast peroxiredoxins displayoxidative stress-dependent switching from a peroxidase to a mo-lecular chaperone function. Cell 117: 625–635.

Jiang, J., K. Prasad, E. M. Lafer, and R. Sousa, 2005 Structuralbasis of interdomain communication in the Hsc70 chaperone.Mol. Cell 20: 513–524.

Jones, R. H., S. Moreno, P. Nurse, and N. C. Jones, 1988 Expressionof the SV40 promoter in fission yeast: identification and charac-terization of an AP-1-like factor. Cell 53: 659–667.

Juhnke, H., C. Charizanis, F. Latifi, and B. Krems, 2000 The es-sential protein fap7 is involved in the oxidative stress responseof Saccharomyces cerevisiae. Mol. Microbiol. 35: 936–948.

Kabani, M., J. M. Beckerich, and C. Gaillardin, 2000 Sls1p stim-ulates Sec63p-mediated activation of Kar2p in a conformation-dependent manner in the yeast endoplasmic reticulum. Mol.Cell. Biol. 20: 6923–6934.

Kabani, M., J. M. Beckerich, and J. L. Brodsky, 2002 Nucleotideexchange factor for the yeast Hsp70 molecular chaperoneSsa1p. Mol. Cell. Biol. 22: 4677–4689.

Kampinga, H. H., and E. A. Craig, 2010 The HSP70 chaperonemachinery: J proteins as drivers of functional specificity. Nat.Rev. Mol. Cell Biol. 11: 579–592.

Kang, S. W., S. G. Rhee, T. S. Chang, W. Jeong, and M. H. Choi,2006 2-Cys peroxiredoxin function in intracellular signaltransduction: therapeutic implications. Trends Mol. Med. 11:571–578.

Kato, K., Y. Yamamoto, and S. Izawa, 2011 Severe ethanol stressinduces assembly of stress granules in Saccharomyces cerevi-siae. Yeast 28: 339–347.

Ketela, T., J. L. Brown, R. C. Stewart, and H. Bussey, 1998 YeastSkn7p activity is modulated by the Sln1p-Ypd1p osmosensorand contributes to regulation of the HOG pathway. Mol. Gen.Genet. 259: 372–378.

Khan, M. A., P. B. Chock, and E. R. Stadtman, 2005 Knockout ofcaspase-like gene, YCA1, abrogates apoptosis and elevates oxi-dized proteins in Saccharomyces cerevisiae. Proc. Natl. Acad.Sci. USA 102: 17326–17331.

Kitagawa, K., D. Skowyra, S. J. Elledge, J. W. Harper, and P. Hieter,1999 SGT1 encodes an essential component of the yeast ki-netochore assembly pathway and a novel subunit of the SCFubiquitin ligase complex. Mol. Cell 4: 21–33.

Koc, A., C. K. Mathews, L. J. Wheeler, M. K. Gross, and G. F. Merrill,2006 Thioredoxin is required for deoxyribonucleotide poolmaintenance during S phase. J. Biol. Chem. 281: 15058–15063.

Kosower, N. S., and E. M. Kosower, 1995 Diamide: an oxidantprobe for thiols. Methods Enzymol. 251: 123–133.

Krasley, E., K. F. Cooper, M. J. Mallory, R. Dunbrack, and R. Strich,2006 Regulation of the oxidative stress response throughSlt2p-dependent destruction of cyclin C in Saccharomyces cere-visiae. Genetics 172: 1477–1486.

Kremer, S. B., and D. S. Gross, 2009 SAGA and Rpd3 chromatinmodification complexes dynamically regulate heat shock genestructure and expression. J. Biol. Chem. 284: 32914–32931.

Krems, B., C. Charizanis and K.-D. Entian, 1996 The responseregulator-like protein Pos9/Skn7 of Saccharomyces cerevisiae isinvolved in oxidative stress resistance. Curr. Genet. 29: 327–334.

Kuchin, S., and M. Carlson, 1998 Functional relationships ofSrb10-Srb11 kinase, carboxy-terminal domain kinase CTDK-I,and transcriptional corepressor Ssn6-Tup1. Mol. Cell. Biol. 18:1163–1171.

Kuge, S., and N. Jones, 1994 YAP1 dependent activation of TRX2is essential for the response of Saccharomyces cerevisiae to oxi-dative stress by hydroperoxides. EMBO J. 13: 655–664.

Kuge, S., N. Jones, and A. Nomoto, 1997 Regulation of yAP-1nuclear localization in response to oxidative stress. EMBO J.16: 1710–1720.

Kuge, S., T. Toda, N. Iizuka, and A. Nomoto, 1998 Crm1 (Xpo1)dependent nuclear export of the budding yeast transcriptionfactor yAP-1 is sensitive to oxidative stress. Genes Cells 3:521–532.

Lallet, S., H. Garreau, C. Poisier, E. Boy-Marcotte, and M. Jacquet,2004 Heat shock-induced degradation of Msn2p, a Saccharo-myces cerevisiae transcription factor, occurs in the nucleus. Mol.Genet. Genomics 272: 353–362.

Laloraya, S., B. D. Gambill, and E. A. Craig, 1994 A role for a eu-karyotic GrpE-related protein, Mge1p, in protein translocation.Proc. Natl. Acad. Sci. USA 91: 6481–6485.

Le, D. T., B. C. Lee, S. M. Marino, Y. Zhang, D. E. Fomenko et al.,2009 Functional analysis of free methionine-R-sulfoxide re-ductase from Saccharomyces cerevisiae. J. Biol. Chem. 284:4354–4364.

Le Moan, N., G. Clement, S. Le Maout, F. Tacnet, and M. B. Toledano,2006 The Saccharomyces cerevisiae proteome of oxidized proteinthiols: contrasted functions for the thioredoxin and glutathionepathways. J. Biol. Chem. 281: 10420–10430.

Lee, D. H., and A. L. Goldberg, 1998 Proteasome inhibitors causeinduction of heat shock proteins and trehalose, which togetherconfer thermotolerance in Saccharomyces cerevisiae. Mol. Cell.Biol. 18: 30–38.

Lee, J., A. Romeo, and D. J. Kosman, 1996 Transcriptional remod-eling and G1 arrest in dioxygen stress in Saccharomyces cerevi-siae. J. Biol. Chem. 271: 24885–24893.

Lee, J., C. Godon, G. Lagniel, D. Spector, J. Garin et al.,1999a Yap1 and Skn7 control two specialized oxidativestress response regulons in yeast. J. Biol. Chem. 181: 16040–16046.

Lee, J., D. Spector, C. Godon, J. Labarre, and M. B. Toledano,1999b A new antioxidant with alkyl hydroperoxide defenseproperties in yeast. J. Biol. Chem. 274: 4537–4544.

Lee, J.-C., M. J. Straffon, T.-Y. Jang, C. M. Grant, and I. W. Dawes,2001 The essential and ancillary role of glutathione in Saccha-romyces cerevisiae: Studies with a grande gsh1 disruptant strain.FEM. Yeast Res. 1: 57–65.

Lee, P., J. Rao, A. Fliss, E. Yang, S. Garrett et al., 2002 The Cdc37protein kinase-binding domain is sufficient for protein kinaseactivity and cell viability. J. Cell Biol. 159: 1051–1059.

Lee, P., B. R. Cho, H. S. Joo, and J. S. Hahn, 2008 Yeast Yak1kinase, a bridge between PKA and stress-responsive transcrip-tion factors, Hsf1 and Msn2/Msn4. Mol. Microbiol. 70: 882–895.

Lee, S., T. Carlson, N. Christian, K. Lea, J. Kedzie et al., 2000 Theyeast heat shock transcription factor changes conformation inresponse to superoxide and temperature. Mol. Biol. Cell 11:1753–1764.

Lenssen, E., U. Oberholzer, J. Labarre, C. De Virgilio, and M. A.Collart, 2002 Saccharomyces cerevisiae Ccr4-not complex


contributes to the control of Msn2p-dependent transcriptionby the Ras/cAMP pathway. Mol. Microbiol. 43: 1023–1037.

Lenssen, E., N. James, I. Pedruzzi, F. Dubouloz, E. Cameroni et al.,2005 The Ccr4-Not complex independently controls bothMsn2-dependent transcriptional activation–via a newly identi-fied Glc7/Bud14 type I protein phosphatase module–and TFIIDpromoter distribution. Mol. Cell. Biol. 25: 488–498.

Lenssen, E., N. Azzouz, A. Michel, E. Landrieux, and M. A. Collart,2007 The Ccr4-not complex regulates Skn7 through Srb10kinase. Eukaryot. Cell 6: 2251–2259.

Leppert, G., R. McDevitt, S. C. Falco, T. K. Van Dyk, M. B. Fickeet al., 1990 Cloning by gene amplification of two loci confer-ring multiple drug resistance in Saccharomyces. Genetics 125:13–20.

Levin, D. E., 2005 Cell wall integrity signaling in Saccharomycescerevisiae. Microbiol. Mol. Biol. Rev. 69: 262–291.

Li, S., A. Ault, C. L. Malone, D. Raitt, S. Dean et al., 1998 Theyeast histidine protein kinase, Sln1p, mediates phosphotransferto two response regulators, Ssk1p and Skn7p. EMBO J. 17:6952–6962.

Li, Z.-S., M. Szczypka, Y.-P. Lu, D. J. Thiele, and P. A. Rea,1996 The yeast cadmium factor protein (YCF1) is a vacuolarglutathione S-conjugate pump. J. Biol. Chem. 271: 6509–6517.

Liochev, S. I., and I. Fridovich, 1999 Superoxide and iron: part-ners in crime. IUBMB Life 48: 157–161.

Lisowsky, T., and A. Meister, 1993 A high copy number of yeastgamma-glutamylcysteine synthetase suppresses a nuclear muta-tion affecting mitochondrial translation. Curr. Genet. 23: 408–413.

Liu, P. C., and D. J. Thiele, 1999 Modulation of human heat shockfactor trimerization by the linker domain. J. Biol. Chem. 274:17219–17225.

Liu, X.-D., and D. J. Thiele, 1996 Oxidative stress induces heatshock factor phosphorylation and HSF-dependent activationof yeast metallothionein gene transcription. Genes Dev. 10:592–603.

Liu, X. D., P. C. Liu, N. Santoro, and D. J. Thiele,1997 Conservation of a stress response: human heat shocktranscription factors functionally substitute for yeast HSF.EMBO J. 16: 6466–6477.

Liu, X. D., K. A. Morano, and D. J. Thiele, 1999 The yeast Hsp110family member, Sse1, is an Hsp90 cochaperone. J. Biol. Chem.274: 26654–26660.

Longo, V. D., E. D. Gralla, and J. S. Valentine,1996 Superoxide dismutase activity is essential for station-ary phase survival in Saccharomyces cerevisae. J. Biol. Chem.271: 12275–12280.

López-Barea, J., J. A. Bárcena, J. A. Bocanegra, J. Florindo, C.García-Alfonso et al., 1990 Structure, mechanism, functions,and regulatory properties of glutathione reductase, pp. 105–116in Glutathione: Metabolism and Physiological Functions, editedby J. Vina. CRC Press, Boca Raton, FL.

López-Mirabal, H. R., and J. R. Winther, 2008 Redox character-istics of the eukaryotic cytosol. Biochim. Biophys. Acta 1783:629–640.

Luikenhuis, S., I. W. Dawes, and C. M. Grant, 1997 The yeastSaccharomyces cerevisiae contains two glutaredoxin genes thatare required for protection against reactive oxygen species. Mol.Biol. Cell 9: 1081–1091.

Machado, A. K., B. A. Morgan, and G. F. Merrill,1997 Thioredoxin reductase-dependent inhibition of MCB cellcycle box activity in Saccharomyces cerevisiae. J. Biol. Chem.272: 17045–17054.

Madeo, F., E. Frohlich, M. Ligr, M. Grey, S. J. Sigrist et al.,1999 Oxygen stress: a regulator of apoptosis in yeast. J. CellBiol. 145: 757–767.

Madeo, F., E. Herker, C. Maldener, S. Wissing, S. Lachelt et al.,2002 A caspase-related protease regulates apoptosis in yeast.Mol. Cell 9: 911–917.

Malik, S., and R. G. Roeder, 2010 The metazoan Mediator co-activator complex as an integrative hub for transcriptional reg-ulation. Nat. Rev. Genet. 11: 761–772.

Mandal, A. K., P. Lee, J. A. Chen, N. Nillegoda, A. Heller et al.,2007 Cdc37 has distinct roles in protein kinase quality controlthat protect nascent chains from degradation and promote post-translational maturation. J. Cell Biol. 176: 319–328.

Martinez-Pastor, M. T., G. Marchler, C. Schuller, A. Marchler-Bauer,H. Ruis et al., 1996 The Saccharomyces cerevisae zinc fingerproteins Msn2p and Msn4p are required for transcriptional in-duction through the stress-response element. EMBO J. 15:2227–2235.

Mascarenhas, M., L. C. Edwards-Ingram, L. Zeef, L. Shenton, M. P.Ashe et al., 2008 Gcn4 is required for the response to peroxidestress in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 19:2995–3007.

Mason, J. T., S. K. Kim, D. B. Knaff, and M. J. Wood,2006 Thermodynamic basis for redox regulation of theYap1 signal transduction pathway. Biochemistry 45: 13409–13417.

Mayordomo, I., F. Estruch, and P. Sanz, 2002 Convergence of thetarget of rapamycin and the Snf1 protein kinase pathways in theregulation of the subcellular localization of Msn2, a transcrip-tional activator of STRE (Stress Response Element)-regulatedgenes. J. Biol. Chem. 277: 35650–35656.

Mazzoni, C., I. D’Addario, and C. Falcone, 2007 The C-terminus ofthe yeast Lsm4p is required for the association to P-bodies. FEBSLett. 581: 4836–4840.

McDaniel, D., A. J. Caplan, M. S. Lee, C. C. Adams, B. R. Fishelet al., 1989 Basal-level expression of the yeast HSP82 generequires a heat shock regulatory element. Mol. Cell. Biol. 9:4789–4798.

Meister, A., 1988 Glutathione metabolism and its selective mod-ification. J. Biol. Chem. 263: 17205–17208.

Mesecke, N., A. Spang, M. Deponte, and J. M. Herrmann, 2008 ANovel Group of Glutaredoxins in the cis-Golgi Critical for Oxi-dative Stress Resistance. Mol. Biol. Cell 19: 2673–2680.

Meyer, Y., W. Siala, T. Bashandy, C. Riondet, F. Vignols et al.,2008 Glutaredoxins and thioredoxins in plants. Biochim. Bio-phys. Acta 1783: 589–600.

Michiels, C., M. Raes, O. Toussaint, and J. Remacle, 1994 Im-portance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radic.Biol. Med. 17: 235–248.

Millson, S. H., A. W. Truman, V. King, C. Prodromou, L. H. Pearlet al., 2005 A two-hybrid screen of the yeast proteome forHsp90 interactors uncovers a novel Hsp90 chaperone require-ment in the activity of a stress-activated mitogen-activated pro-tein kinase, Slt2p (Mpk1p). Eukaryot. Cell 4: 849–860.

Molina, M. M., G. Belli, M. A. de la Torre, M. T. Rodriguez-Manzaneque, and E. Herrero, 2004 Nuclear monothiolglutaredoxins of Saccharomyces cerevisiae can function as mito-chondrial glutaredoxins. J. Biol. Chem. 279: 51923–51930.

Molina-Navarro, M. M., L. Castells-Roca, G. Belli, J. Garcia-Martinez,J. Marin-Navarro et al., 2008 Comprehensive transcriptionalanalysis of the oxidative response in yeast. J. Biol. Chem. 283:17908–17918.

Mollapour, M., S. Tsutsumi, A. C. Donnelly, K. Beebe, M. J. Tokitaet al., 2010 Swe1Wee1-dependent tyrosine phosphorylation ofHsp90 regulates distinct facets of chaperone function. Mol. Cell37: 333–343.

Monteiro, G., B. B. Horta, D. C. Pimenta, O. Augusto, and L. E.Netto, 2007 Reduction of 1-Cys peroxiredoxins by ascorbatechanges the thiol-specific antioxidant paradigm, revealing


another function of vitamin C. Proc. Natl. Acad. Sci. USA 104:4886–4891.

Morano, K. A., N. Santoro, K. A. Kock, and D. J. Thiele, 1999 Atrans-activation domain in yeast heat shock transcription factoris essential for cell cycle progression during stress. Mol. Cell.Biol. 19: 402–411.

Morgan, B. A., and E. A. Veal, 2007 Functions of typical 2-Cysperoxiredoxins in yeast. Subcell. Biochem. 44: 253–265.

Morgan, B. A., G. R. Banks, W. M. Toone, D. Raitt, S. Kuge et al.,1997 The Skn7 response regulator controls gene expression inthe oxidative stress response of the budding yeast Saccharomycescerevisiae. EMBO J. 16: 1035–1044.

Morimoto, R. I., 2008 Proteotoxic stress and inducible chaperonenetworks in neurodegenerative disease and aging. Genes Dev.22: 1427–1438.

Mosser, D. D., S. Ho, and J. R. Glover, 2004 Saccharomyces cer-evisiae Hsp104 enhances the chaperone capacity of human cellsand inhibits heat stress-induced proapoptotic signaling. Bio-chemistry 43: 8107–8115.

Muhlenhoff, U., S. Molik, J. R. Godoy, M. A. Uzarska, N. Richteret al., 2010 Cytosolic monothiol glutaredoxins function inintracellular iron sensing and trafficking via their bound iron-sulfur cluster. Cell Metab. 12: 373–385.

Mukai, H., T. Kuno, H. Tanaka, D. Hirata, T. Miyakawa et al.,1993 Isolation and characterization of SSE1 and SSE2, newmembers of the yeast HSP70 multigene family. Gene 132:57–66.

Mukhopadhyay, R., J. Shi, and B. P. Rosen, 2000 Purification andcharacterization of ACR2p, the Saccharomyces cerevisiae arse-nate reductase. J. Biol. Chem. 275: 21149–21157.

Mulder, K. W., A. Inagaki, E. Cameroni, F. Mousson, G. S. Winkleret al., 2007 Modulation of Ubc4p/Ubc5p-mediated stress re-sponses by the RING-finger-dependent ubiquitin-protein ligaseNot4p in Saccharomyces cerevisiae. Genetics 176: 181–192.

Muller, E. G. D., 1991 Thioredoxin deficiency in yeast prolongsS phase and shortens the G1 interval of the cell cycle. J. Biol.Chem. 266: 9194–9202.

Muller, E. G. D., 1996 A glutathione reductase mutant of yeastaccumulates high levels of oxidized glutathione and requiresthioredoxin for growth. Mol. Biol. Cell 7: 1805–1813.

Murphy, M. P., 2009 How mitochondria produce reactive oxygenspecies. Biochem. J. 417: 1–13.

Natarajan, K., M. R. Meyer, B. M. Jackson, D. Slade, C. Robertset al., 2001 Transcriptional profiling shows that Gcn4p isa master regulator of gene expression during amino acid star-vation in yeast. Mol. Cell. Biol. 21: 4347–4368.

Nathan, D. F., M. H. Vos, and S. Lindquist, 1997 In vivo functionsof the Saccharomyces cerevisiae Hsp90 chaperone. Proc. Natl.Acad. Sci. USA 94: 12949–12956.

Neef, D. W., M. L. Turski, and D. J. Thiele, 2010 Modulation ofheat shock transcription factor 1 as a therapeutic target for smallmolecule intervention in neurodegenerative disease. PLoS Biol.8: e1000291.

Nelson, R. J., T. Ziegelhoffer, C. Nicolet, M. Werner-Washburne,and E. A. Craig, 1992 The translation machinery and 70 kdheat shock protein cooperate in protein synthesis. Cell 71: 97–105.

Nicholls, S., M. Straffon, B. Enjalbert, A. Nantel, S. Macaskill et al.,2004 Msn2- and Msn4-like transcription factors play no obvi-ous roles in the stress responses of the fungal pathogen Candidaalbicans. Eukaryot. Cell 3: 1111–1123.

Nicholls, S., M. D. Leach, C. L. Priest, and A. J. Brown, 2009 Roleof the heat shock transcription factor, Hsf1, in a major fungalpathogen that is obligately associated with warm-blooded ani-mals. Mol. Microbiol. 74: 844–861.

Nicholls, S., D. M. Maccallum, F. A. Kaffarnik, L. Selway, S. C. Pecket al., 2010 Activation of the heat shock transcription factor Hsf1

is essential for the full virulence of the fungal pathogen Candidaalbicans. Fungal Genet. Biol. 48: 297–305.

Nunes, E., and W. Siede, 1996 Hyperthermia and paraquat-induced G1 arrest in the yeast Saccharomyces cerevisiae isindependent of the RAD9 gene. Radiat. Environ. Biophys.35: 55–57.

Ohdate, T., K. Kita, and Y. Inoue, 2010 Kinetics and redox regu-lation of Gpx1, an atypical 2-Cys peroxiredoxin, in Saccharomy-ces cerevisiae. FEM. Yeast Res. 10: 787–790.

Ohkuni, K., R. Abdulle, A. H. Tong, C. Boone and K. Kitagawa,2008 Ybp2 associates with the central kinetochore of Saccha-romyces cerevisiae and mediates proper mitotic progression.PLoS One 3: e1617.

Ohtake, Y., and S. Yabuuchi, 1991 Molecular cloning of thegamma-glutamylcysteine synthetase gene of Saccharomycescerevisiae. Yeast 7: 953–961.

Okazaki, S., A. Naganuma, and S. Kuge, 2005 Peroxiredoxin-mediated redox regulation of the nuclear localization of Yap1,a transcription factor in budding yeast. Antioxid. Redox Signal.7: 327–334.

Okazaki, S., T. Tachibana, A. Naganuma, N. Mano, and S. Kuge,2007 Multistep disulfide bond formation in Yap1 is requiredfor sensing and transduction of H2O2 stress signal. Mol. Cell 27:675–688.

Page, N., J. Sheraton, J. L. Brown, R. C. Stewart, and H. Bussey,1996 Identification of ASK10 as a multicopy activator ofSkn7p-dependent transcription of a HIS3 reporter gene. Yeast12: 267–272.

Palmer, L. K., J. L. Shoemaker, B. A. Baptiste, D. Wolfe, and R. L.Keil, 2005 Inhibition of translation initiation by volatileanesthetics involves nutrient-sensitive GCN-independent and-dependent processes in yeast. Mol. Biol. Cell 16: 3727–3739.

Panaretou, B., G. Siligardi, P. Meyer, A. Maloney, J. K. Sullivanet al., 2002 Activation of the ATPase activity of hsp90 by thestress-regulated cochaperone aha1. Mol. Cell 10: 1307–1318.

Park, S. G., M.-K. Cha, W. Jeong, and I.-H. Kim, 2000 Distinctphysiological functions of thiol peroxidase isoenzymes in Sac-charomyces cerevisiae. J. Biol. Chem. 275: 5723–5732.

Parker, R., and U. Sheth, 2007 P bodies and the control of mRNAtranslation and degradation. Mol. Cell 25: 635–646.

Parsell, D. A., A. S. Kowal, M. A. Singer, and S. Lindquist,1994 Protein disaggregation mediated by heat-shock proteinHsp104. Nature 372: 475–478.

Patel, J., L. E. McLeod, R. G. Vries, A. Flynn, X. Wang et al.,2002 Cellular stresses profoundly inhibit protein synthesisand modulate the states of phosphorylation of multiple trans-lation factors. Eur. J. Biochem. 269: 3076–3085.

Pavitt, G. D., K. V. Ramaiah, S. R. Kimball, and A. G. Hinnebusch,1998 eIF2 independently binds two distinct eIF2B subcom-plexes that catalyze and regulate guanine-nucleotide exchange.Genes Dev. 12: 514–526.

Pedrajas, J. R., E. Kosmidou, A. Miranda-Vizuete, J.-A. Gustafsson,A. P. H. Wright et al., 1999 Identification and functional char-acterization of a novel mitochondrial thioredoxin system in Sac-charomyces cerevisiae. J. Biol. Chem. 274: 6366–6373.

Pedrajas, J. R., A. Miranda-Vizuete, N. Javanmardy, J.-A. Gustafsson,and G. Spyrou, 2000 Mitochondria of Saccharomyces cerevi-siae contain one-conserved cysteine type peroxiredoxin withthioredoxin peroxidase activity. J. Biol. Chem. 275: 16296–16301.

Pedrajas, J. R., C. A. Padilla, B. N. McDonagh, and J. A. Bárcena,2010 Glutaredoxin participates in the reduction of peroxidesby the mitochondrial 1-CYS peroxiredoxin in Saccharomyces cer-evisiae. Antioxid. Redox Signal. 13: 249–258.

Pereira, C., R. D. Silva, L. Saraiva, B. Johansson, M. J. Sousa et al.,2008 Mitochondria-dependent apoptosis in yeast. Biochim.Biophys. Acta 1783: 1286–1302.


Perrone, G. G., S. X. Tan, and I. W. Dawes, 2008 Reactive oxygenspecies and yeast apoptosis. Biochim. Biophys. Acta 1783:1354–1368.

Picard, D., Picard Laboratory. Available at: http://www.picard.ch/downloads. Accessed: November 2011.

Piper, P. W., 1997 The yeast heat shock response, pp. 75–99 inYeast Stress Responses, edited by S. Hohmann, and W. E. Mager.Chapman and Hall, New York.

Polier, S., Z. Dragovic, F. U. Hartl, and A. Bracher, 2008 Structuralbasis for the cooperation of Hsp70 and Hsp110 chaperones inprotein folding. Cell 133: 1068–1079.

Pollegioni, L., L. Piubelli, S. Sacchi, M. S. Pilone, and G. Molla,2007 Physiological functions of D-amino acid oxidases: fromyeast to humans. Cell. Mol. Life Sci. 64: 1373–1394.

Preiss, T., J. Baron-Benhamou, W. Ansorge, and M. W. Hentze,2003 Homodirectional changes in transcriptome compositionand mRNA translation induced by rapamycin and heat shock.Nat. Struct. Biol. 10: 1039–1047.

Quan, X., R. Rassadi, B. Rabie, N. Matusiewicz, and U. Stochaj,2004 Regulated nuclear accumulation of the yeast hsp70Ssa4p in ethanol-stressed cells is mediated by the N-terminaldomain, requires the nuclear carrier Nmd5p and protein kinaseC. FASEB J. 18: 899–901.

Raitt, D. C., A. L. Johnson, A. M. Erkine, K. Makino, B. A. Morganet al., 2000 The Skn7 response regulator of Saccharomycescerevisiae interacts with Hsf1 in vivo and is required for theinduction of heat shock genes by oxidative stress. Mol. Biol. Cell11: 2335–2347.

Ralser, M., G. Heeren, M. Breitenbach, H. Lehrach, and S. Krobitsch,2006 Triose phosphate isomerase deficiency is caused by altereddimerization–not catalytic inactivity–of the mutant enzymes. PLoSONE 1: e30.

Ralser, M., M. M. Walmelink, A. Kowald, B. Gerisch, G. Heerenet al., 2007 Dynamic re-routing of the carbohydrate flux iskey to counteracting oxidative stress. J. Biol. 6: 10.

Ralser, M., M. M. Wamelink, S. Latkolik, E. E. Jansen, H. Lehrachet al., 2009 Metabolic reconfiguration precedes transcriptionalregulation in the antioxidant response. Nat. Biotechnol. 27:604–605.

Rand, J. D., and C. M. Grant, 2006 The thioredoxin system pro-tects ribosomes against stress-induced aggregation. Mol. Biol.Cell 17: 387–401.

Raviol, H., H. Sadlish, F. Rodriguez, M. P. Mayer, and B. Bukau,2006 Chaperone network in the yeast cytosol: Hsp110 is re-vealed as an Hsp70 nucleotide exchange factor. EMBO J. 25:2510–2518.

Rhee, S. G., S. W. Kang, W. Jeong, T. S. Chang, K. S. Yang et al.,2005 Intracellular messenger function of hydrogen peroxideand its regulation by peroxiredoxins. Curr. Opin. Cell Biol. 17:183–189.

Rodrigues-Pousada, C., R. A. Menezes, and C. Pimentel,2010 The Yap family and its role in stress response. Yeast27: 245–258.

Rodriguez-Manzaneque, M. T., J. Ros, E. Cabiscol, A. Sorribas, andE. Herrero, 1999 Grx5 glutaredoxin plays a central role in pro-tection against protein oxidative damage in Saccharomyces cer-evisiae. Mol. Cell. Biol. 19: 8180–8190.

Rodriguez-Manzaneque, M. T., J. Tamarit, G. Belli, J. Ros, and E.Herrero, 2002 Grx5 is a mitochondrial glutaredoxin requiredfor the activity of iron/sulfur enzymes. Mol. Biol. Cell 13: 1109–1121.

Roveri, A., M. Maiorino, and F. Ursini, 1994 Enzymatic and im-munological measurements of soluble and membrane-boundphospholipid-hydroperoxide glutathione peroxidases. MethodsEnzymol. 233: 202–212.

Rowley, A., G. C. Johnston, B. Butler, M. Werner-Washburne, andR. A. Singer, 1993 Heat shock-mediated cell cycle blockage

and G1 cyclin expression in the yeast Saccharomyces cerevisiae.Mol. Cell. Biol. 13: 1034–1041.

Ruiz-Roig, C., C. Vieitez, F. Posas, and E. de Nadal, 2010 TheRpd3L HDAC complex is essential for the heat stress responsein yeast. Mol. Microbiol. 76: 1049–1062.

Saavedra, C., K. S. Tung, D. C. Amberg, A. K. Hopper, and C. N.Cole, 1996 Regulation of mRNA export in response to stress inSaccharomyces cerevisiae. Genes Dev. 10: 1608–1620.

Saavedra, C. A., C. M. Hammell, C. V. Heath, and C. N. Cole,1997 Yeast heat-shock mRNAs are exported through a distinctpathway defined by Rip1p. Genes Dev. 11: 2845–2856.

Sanchez, Y., and S. L. Lindquist, 1990 HSP104 required for in-duced thermotolerance. Science 248: 1112–1115.

Santoro, N., N. Johansson, and D. J. Thiele, 1998 Heat shockelement architecture is an important determinant in the temper-ature and transactivation domain requirements for heat shocktranscription factor. Mol. Cell. Biol. 18: 6340–6352.

Saris, N., H. Holkeri, R. A. Craven, C. J. Stirling, and M. Makarow,1997 The Hsp70 homologue Lhs1p is involved in a novel func-tion of the yeast endoplasmic reticulum, refolding and stabilizationof heat-denatured protein aggregates. J. Cell Biol. 137: 813–824.

Schafer, F. Q., and G. R. Buettner, 2001 Redox environment of thecell as viewed through the redox state of the glutathione disul-fide/glutathione couple. Free Radic. Biol. Med. 30: 1191–1212.

Schmitt, A. P., and K. McEntee, 1996 Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistressresponse in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci.USA 93: 5777–5782.

Schnell, N., and K. D. Entian, 1991 Identification and character-ization of a Saccharomyces cerevisiae gene (PAR1) conferringresistance to iron chelators. Eur. J. Biochem. 200: 487–493.

Schnell, N., B. Krems, and K.-D. Entian, 1992 The PAR1 (YAP1/SNQ3) gene of Saccharomyces cerevisiae, a c-jun homolog, isinvolved in oxygen metabolism. Curr. Genet. 21: 269–273.

Schuermann, J. P., J. Jiang, J. Cuellar, O. Llorca, L. Wang et al.,2008 Structure of the Hsp110:Hsc70 nucleotide exchange ma-chine. Mol. Cell 31: 232–243.

Shackelford, R. E., W. K. Kaufmann, and R. S. Paules,2000 Oxidative stress and cell cycle checkpoint function. FreeRadic. Biol. Med. 28: 1387–1404.

Shaner, L., and K. A. Morano, 2007 All in the family: atypicalHsp70 chaperones are conserved modulators of Hsp70 activity.Cell Stress Chaperones 12: 1–8.

Shaner, L., H. Wegele, J. Buchner, and K. A. Morano, 2005 Theyeast Hsp110 Sse1 functionally interacts with the Hsp70 chap-erones Ssa and Ssb. J. Biol. Chem. 280: 41262–41269.

Shaner, L., R. Sousa, and K. A. Morano, 2006 Characterization ofhsp70 binding and nucleotide exchange by the yeast hsp110chaperone sse1. Biochemistry 45: 15075–15084.

Shaner, L., P. A. Gibney, and K. A. Morano, 2008 The Hsp110protein chaperone Sse1 is required for yeast cell wall integrityand morphogenesis. Curr. Genet. 54: 1–11.

Shanmuganathan, A., S. V. Avery, S. A. Willetts, and J. E.Houghton, 2004 Copper-induced oxidative stress in Saccharo-myces cerevisiae targets enzymes of the glycolytic pathway.FEBS Lett. 556: 253–259.

Sheehan, D., G. Meade, V. M. Foley, and C. A. Dowd,2001 Structure, function and evolution of glutathione trans-ferases: implications for classification of non-mammalian mem-bers of an ancient enzyme superfamily. Biochem. J. 360: 1–16.

Shenton, D., and C. M. Grant, 2003 Protein S-thiolation targetsglycolysis and protein synthesis in response to oxidative stress inthe yeast Saccharomyces cerevisiae. Biochem. J. 374: 513–519.

Shenton, D., J. B. Smirnova, J. N. Selley, K. Carroll, S. J. Hubbardet al., 2006 Global translational responses to oxidative stressimpact upon multiple levels of protein synthesis. J. Biol. Chem.281: 29011–29021.




Shi, Z. Z., J. Osei-Frimpong, G. Kala, S. V. Kala, R. J. Barrios et al.,2000 Glutathione synthesis is essential for mouse develop-ment but not for cell growth in culture. Proc. Natl. Acad. Sci.USA 97: 5101–5106.

Shivaswamy, S., and V. R. Iyer, 2008 Stress-dependent dynamics ofglobal chromatin remodeling in yeast: dual role for SWI/SNF inthe heat shock stress response. Mol. Cell. Biol. 28: 2221–2234.

Simic, M. G., D. S. Bergtold, and L. R. Karam, 1989 Generation ofoxy radicals in biosystems. Mutat. Res. 214: 3–12.

Singer, M. A., and S. Lindquist, 1998 Thermotolerance in Saccha-romyces cerevisiae: the Yin and Yang of trehalose. Trends Bio-technol. 16: 460–468.

Singh, S. D., N. Robbins, A. K. Zaas, W. A. Schell, J. R. Perfect et al.,2009 Hsp90 governs echinocandin resistance in the patho-genic yeast Candida albicans via calcineurin. PLoS Pathog. 5:e1000532.

Slekar, K. H., D. J. Kosman, and V. C. Culotta, 1996 The yeastcopper/zinc superoxide dismutase and the pentose phosphatepathway play overlapping roles in oxidative stress protection.J. Biol. Chem. 271: 28831–28836.

Smirnova, J. B., J. N. Selley, F. Sanchez-Cabo, K. Carroll, A. A. Eddyet al., 2005 Global Gene Expression Profiling Reveals Wide-spread yet Distinctive Translational Responses to Different Eu-karyotic Translation Initiation Factor 2B-Targeting StressPathways. Mol. Cell. Biol. 25: 9340–9349.

Smith, A., M. P. Ward, and S. Garrett, 1998 Yeast PKA repressesMsn2p/Msn4p-dependent gene expression to regulate growth,stress response and glycogen accumulation. EMBO J. 17: 3556–3564.

Sondermann, H., A. K. Ho, L. L. Listenberger, K. Siegers, I. Moarefiet al., 2002 Prediction of novel Bag-1 homologs based onstructure/function analysis identifies Snl1p as an Hsp70 co-chaperone in Saccharomyces cerevisiae. J. Biol. Chem. 277:33220–33227.

Sorger, P. K., 1990 Yeast heat shock factor contains separabletransient and sustained response transcriptional activators. Cell62: 793–805.

Sorger, P. K., and H. R. Pelham, 1987 Purification and character-ization of a heat-shock element binding protein from yeast.EMBO J. 6: 3035–3041.

Spector, D., J. Labarre, and M. B. Toledano, 2001 A genetic in-vestigation of the role of glutathione: mutations in the prolinebiosynthesis pathway are the only suppressors of glutathioneauxotrophy in yeast. J. Biol. Chem. 276: 7011–7016.

Spickett, C. M., N. Smirnoff, and A. R. Pitt, 2000 The biosynthesisof erythroascorbate in Saccharomyces cerevisiae and its role asan antioxidant. Free Radic. Biol. Med. 28: 183–192.

Stadtman, E. R., and R. L. Levine, 2003 Free radical-mediatedoxidation of free amino acids and amino acid residues in pro-teins. Amino Acids 25: 207–218.

Stadtman, E. R., J. Moskovitz, and R. L. Levine, 2003 Oxidation ofmethionine residues of proteins: biological consequences. Anti-oxid. Redox Signal. 5: 577–582.

Steel, G. J., D. M. Fullerton, J. R. Tyson, and C. J. Stirling,2004 Coordinated activation of Hsp70 chaperones. Science303: 98–101.

Stromer, T., E. Fischer, K. Richter, M. Haslbeck, and J. Buchner,2004 Analysis of the regulation of the molecular chaperoneHsp26 by temperature-induced dissociation: the N-terminal do-mail is important for oligomer assembly and the binding of un-folding proteins. J. Biol. Chem. 279: 11222–11228.

Sturtz, L. A., K. Diekert, L. T. Jensen, R. Lill, and V. C. Culotta,2001 A fraction of yeast Cu,Zn-superoxide dismutase and itsmetallochaperone, CCS, localize to the intermembrane space ofmitochondria. A physiological role for SOD1 in guarding againstmitochondrial oxidative damage. J. Biol. Chem. 276: 38084–38089.

Subjeck, J. R., J. J. Sciandra, and R. J. Johnson, 1982 Heat shockproteins and thermotolerance; a comparison of induction kinet-ics. Br. J. Radiol. 55: 579–584.

Sugiyama, K., S. Izawa, and Y. Inoue, 2000a The Yap1p-depen-dent induction of glutathione synthesis in heat shock responseof Saccharomyces cerevisiae. J. Biol. Chem. 275: 15535–15540.

Sugiyama, K., A. Kawamura, S. Izawa, and Y. Inoue, 2000b Roleof glutathione in heat-shock-induced cell death of Saccharomy-ces cerevisiae. Biochem. J. 352(Pt 1): 71–78.

Swaminathan, S., T. Masek, C. Molin, M. Pospisek, and P. Sunner-hagen, 2006 Rck2 Is Required for Reprogramming of Ribo-somes during Oxidative Stress. Mol. Biol. Cell 17: 1472–1482.

Tachibana, T., S. Okazaki, A. Murayama, A. Naganuma, A. Nomotoet al., 2009 A major peroxiredoxin-induced activation of Yap1transcription factor is mediated by reduction-sensitive disulfidebonds and reveals a low level of transcriptional activation.J. Biol. Chem. 284: 4464–4472.

Taipale, M., D. F. Jarosz, and S. Lindquist, 2010 HSP90 at the hubof protein homeostasis: emerging mechanistic insights. Nat. Rev.Mol. Cell Biol. 11: 515–528.

Takahashi, T., T. Yano, J. Zhu, G. W. Hwang, and A. Naganuma,2010 Overexpression of FAP7, MIG3, TMA19, or YLR392cconfers resistance to arsenite on Saccharomyces cerevisiae.J. Toxicol. Sci. 35: 945–946.

Takemori, Y., A. Sakaguchi, S. Matsuda, Y. Mizukami, and H. Sa-kurai, 2006 Stress-induced transcription of the endoplasmicreticulum oxidoreductin gene ERO1 in the yeast Saccharomycescerevisiae. Mol. Genet. Genomics 275: 89–96.

Takemori, Y., Y. Enoki, N. Yamamoto, Y. Fukai, K. Adachi et al.,2009 Mutational analysis of human heat-shock transcriptionfactor 1 reveals a regulatory role for oligomerization in DNA-binding specificity. Biochem. J. 424: 253–261.

Tamai, K. T., X. Liu, P. Silar, T. Sosinowski, and D. J. Thiele,1994 Heat shock transcription factor activates yeast metallo-thionein gene expression in response to heat and glucose star-vation via distinct signalling pathways. Mol. Cell. Biol. 14:8155–8165.

Tan, S. X., D. Greetham, S. Raeth, C. M. Grant, I. W. Dawes et al.,2010 The thioredoxin-thioredoxin reductase system canfunction in vivo as an alternative system to reduce oxidizedglutathione in Saccharomyces cerevisiae. J. Biol. Chem. 285:6118–6126.

Tanaka, T., S. Izawa, and Y. Inoue, 2005 GPX2, encoding aphospholipid hydroperoxide glutathione peroxidase homologue,codes for an atypical 2-Cys peroxiredoxin in Saccharomyces cer-evisiae. J. Biol. Chem. 23: 42078–42087.

Tapia, H., and K. A. Morano, 2010 Hsp90 nuclear accumulation inquiescence is linked to chaperone function and spore develop-ment in yeast. Mol. Biol. Cell 21: 63–72.

Teixeira, D., U. Sheth, M. A. Valencia-Sanchez, M. Brengues, and R.Parker, 2005 Processing bodies require RNA for assembly andcontain nontranslating mRNAs. RNA 11: 371–382.

Temple, M. D., G. G. Perrone, and I. W. Dawes, 2005 Complexcellular responses to reactive oxygen species. Trends Cell Biol.15: 319–326.

Thevelein, J. M., L. Cauwenberg, S. Colombo, J. H. De Winde, M.Donation et al., 2000 Nutrient-induced signal transductionthrough the protein kinase A pathway and its role in the controlof metabolism, stress resistance, and growth in yeast. EnzymeMicrob. Technol. 26: 819–825.

Thorpe, G. W., C. S. Fong, N. Alic, V. J. Higgins, and I. W. Dawes,2004 Cells have distinct mechanisms to maintain protectionagainst different reactive oxygen species: oxidative-stress-response genes. Proc. Natl. Acad. Sci. USA 10: 6564–6569.

Thorsen, M., G. G. Perrone, E. Kristiansson, M. Traini, T. Ye et al.,2009 Genetic basis of arsenite and cadmium tolerance in Sac-charomyces cerevisiae. BMC Genomics 10: 105.


Toda, T., M. Shimanuki, and M. Yanagida, 1991 Fission yeastgenes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to themammalian ERK1/MAP2 and budding yeast FUS3 and KSS1kinases. Genes Dev. 5: 60–73.

Toda, T., M. Shimanuki, Y. Saka, H. Yamano, Y. Adachi et al.,1992 Fission yeast pap1-dependent transcription is negativelyregulated by an essential nuclear protein, crm1. Mol. Cell. Biol.12: 5474–5484.

Toledano, M. B., C. Kumar, N. Le Moan, D. Spector, and F. Tacnet,2007 The system biology of thiol redox system in Escherichiacoli and yeast: differential functions in oxidative stress, ironmetabolism and DNA synthesis. FEBS Lett. 581: 3598–3607.

Toone, W. M., S. Kuge, M. Samuels, B. A. Morgan, T. Toda et al.,1998 Regulation of the fission yeast transcription factor Pap1by oxidative stress: requirement for the nuclear export factorCrm1 (Exportin) and the stress-activated MAP kinase Sty1/Spc1. Genes Dev. 12: 1453–1463.

Trott, A., and K. A. Morano, 2003 The yeast response to heatshock. Yeast Stress Responses 1: 71–119.

Trott, A., J. D. West, L. Klaic, S. D. Westerheide, R. B. Silvermanet al., 2008 Activation of Heat Shock and Antioxidant Responsesby the Natural Product Celastrol: Transcriptional Signatures ofa Thiol-targeted Molecule. Mol. Biol. Cell 19: 1104–1112.

Trotter, E. W., and C. M. Grant, 2003 Non-reciprocal regulation ofthe redox state of the glutathione/glutaredoxin and thioredoxinsystems. EMBO Rep. 4: 184–189.

Trotter, E. W., and C. M. Grant, 2005 Overlapping roles of thecytoplasmic and mitochondrial redoc regulatory systems in theyeast Saccharomyces cerevisiae. Eukaryot. Cell 4: 392–400.

Trotter, E. W., L. Berenfeld, S. A. Krause, G. A. Petsko, and J. V.Gray, 2001 Protein misfolding and temperature up-shift causeG1 arrest via a common mechanism dependent on heat shockfactor in Saccharomycescerevisiae. Proc. Natl. Acad. Sci. USA98: 7313–7318.

Trotter, E. W., C. M. Kao, L. Berenfeld, D. Botstein, G. A. Petskoet al., 2002 Misfolded proteins are competent to mediate a sub-set of the responses to heat shock in Saccharomyces cerevisiae.J. Biol. Chem. 277: 44817–44825.

Truman, A. W., S. H. Millson, J. M. Nuttall, M. Mollapour, C.Prodromou et al., 2007 In the yeast heat shock response,Hsf1-directed induction of Hsp90 facilitates the activation ofthe Slt2 (Mpk1) mitogen-activated protein kinase requiredfor cell integrity. Eukaryot. Cell 6: 744–752.

Tu, B. P., and J. S. Weissman, 2004 Oxidative protein folding ineukaryotes: mechanisms and consequences. J. Cell Biol. 164:341–346.

Turton, H. E., I. W. Dawes, and C. M. Grant, 1997 Saccharomycescerevisiae exhibits a yAP-1-mediated adaptive response to ma-londialdehyde. J. Bacteriol. 179: 1096–1101.

van Loon, A. P. G. M., B. Pesold-Hurt, and G. Schatz, 1986 A yeastmutant lacking mitochondrial manganese-superoxide dismutaseis hypersensitive to oxygen. Proc. Natl. Acad. Sci. USA 83:3820–3824.

Veal, E. A., S. J. Ross, P. Malakasi, E. Peacock, and B. A. Morgan,2003 Ybp1 is required for the hydrogen peroxide-induced ox-idation of the Yap1 transcription factor. J. Biol. Chem. 278:30896–30904.

Veal, E. A., A. M. Day, and B. A. Morgan, 2007 Hydrogen peroxidesensing and signaling. Mol. Cell 26: 1–14.

Vincent, O., S. Kuchin, S. P. Hong, R. Townley, V. K. Vyas et al.,2001 Interaction of the Srb10 kinase with Sip4, a transcrip-tional activator of gluconeogenic genes in Saccharomyces cere-visiae. Mol. Cell. Biol. 21: 5790–5796.

Vogel, J. P., L. M. Misra, and M. D. Rose, 1990 Loss of BiP/GRP78function blocks translocation of secretory proteins in yeast.J. Cell Biol. 110: 1885–1895.

Vogel, M., B. Bukau, and M. P. Mayer, 2006a Allosteric regulationof Hsp70 chaperones by a proline switch. Mol. Cell 21: 359–367.

Vogel, M., M. P. Mayer, and B. Bukau, 2006b Allosteric regulationof Hsp70 chaperones involves a conserved interdomain linker.J. Biol. Chem. 281: 38705–38711.

Voos, W., B. D. Gambill, S. Laloraya, D. Ang, E. A. Craig et al.,1994 Mitochondrial GrpE is present in a complex with hsp70and preproteins in transit across membranes. Mol. Cell. Biol. 14:6627–6634.

Wallace, M. A., L. L. Liou, J. Martins, M. H. Clement, S. Bailey et al.,2004 Superoxide inhibits 4Fe-4S cluster enzymes involved inamino acid biosynthesis. Cross-compartment protection byCuZn-superoxide dismutase. J. Biol. Chem. 279: 32055–32062.

Wandinger, S. K., M. H. Suhre, H. Wegele, and J. Buchner,2006 The phosphatase Ppt1 is a dedicated regulator of themolecular chaperone Hsp90. EMBO J. 25: 367–376.

Wang, L., G. Renault, H. Garreau, and M. Jacquet, 2004 Stressinduces depletion of Cdc25p and decreases the cAMP producingcapability in Saccharomyces cerevisiae. Microbiology 150:3383–3391.

Welker, S., B. Rudolph, E. Frenzel, F. Hagn, G. Liebisch et al.,2010 Hsp12 is an intrinsically unstructured stress protein thatfolds upon membrane association and modulates membranefunction. Mol. Cell 39: 507–520.

Wemmie, J. A., S. M. Steggerda, and W. S. Moye-Rowley,1997 The Saccharomyces cerevisiae AP-1 protein discriminatesbetween oxidative stress elicited by the oxidants H2O2 and di-amide. J. Biol. Chem. 272: 7908–7914.

Werner-Washburne, M., D. E. Stone, and E. A. Craig,1987 Complex interactions among members of an essentialsubfamily of hsp70 genes in Saccharomyces cerevisiae. Mol.Cell. Biol. 7: 2568–2577.

Westerheide, S. D., and R. I. Morimoto, 2005 Heat shock responsemodulators as therapeutic tools for diseases of protein confor-mation. J. Biol. Chem. 280: 33097–33100.

Westerheide, S. D., J. Anckar, S. M. Stevens, Jr., L. Sistonen, and R.I. Morimoto, 2009 Stress-inducible regulation of heat shockfactor 1 by the deacetylase SIRT1. Science 323: 1063–1066.

Wheeler, G. L., K. A. Quinn, G. Perrone, I. W. Dawes, and C. M.Grant, 2002 Glutathione regulates the expression of gamma-glutamylcysteine synthetase via the Met4 transcription factor.Mol. Microbiol. 46: 545–556.

Wheeler, G. L., E. W. Trotter, I. W. Dawes, and C. M. Grant,2003 Coupling of the transcriptional regulation of glutathionebiosynthesis to the availability of glutathione and methioninevia the Met4 and Yap1 transcription factors. J. Biol. Chem. 278:49920–49928.

Wieser, R., G. Adam, A. Wagner, C. Schuller, G. Marchler et al.,1991 Heat shock factor-independent heat control of transcrip-tion of the CTT1 gene encoding the cytosolic catalase T of Sac-charomyces cerevisiae. J. Biol. Chem. 266: 12406–12411.

Winkler, B. S., S. M. Orselli, and T. S. Rex, 1994 The redox couplebetween glutathione and ascorbic acid: a chemical and physio-logical perspective. Free Radic. Biol. Med. 17: 333–349.

Wissing, S., P. Ludovico, E. Herker, S. Buttner, S. M. Engelhardtet al., 2004 An AIF orthologue regulates apoptosis in yeast.J. Cell Biol. 166: 969–974.

Wong, C. M., K. L. Siu, and D. Y. Jin, 2004 Peroxiredoxin-nullyeast cells are hypersensitive to oxidative stress and are genomi-cally unstable. J. Biol. Chem. 22: 23207–23213.

Wood, M. J., G. Storz, and N. Tjandra, 2004 Structural basis forredox regulation of Yap1 transcription factor localization. Na-ture 430: 917–921.

Wood, Z. A., E. Schroder, J. R. Harris, and L. B. Poole,2003 Structure, mechanism and regulation of peroxiredoxins.Trends Biochem. Sci. 28: 32–40.


Wotton, D., K. Freeman, and D. Shore, 1996 Multimerization ofHsp42p, a novel heat shock protein of Saccharomyces cerevi-siae, is dependent on a conserved carboxyl-terminal sequence.J. Biol. Chem. 271: 2717–2723.

Wu, A., and W. S. Moye-Rowley, 1994 GSH1, which encodesg-glutamylcysteine synthetase, is a target gene for yAP-1 tran-scriptional regulation. Mol. Cell. Biol. 14: 5832–5839.

Wu, A., J. A. Wemmie, N. P. Edgington, M. Goebl, J. L. Guevaraet al., 1993 Yeast bZIP proteins mediate pleiotropic drug andmetal resistance. J. Biol. Chem. 268: 18850–18858.

Wysocki, R., and M. J. Tamas, 2010 How Saccharomyces cerevi-siae copes with toxic metals and metalloids. FEMS Microbiol.Rev. 34: 925–951.

Yam, A. Y., V. Albanese, H. T. Lin, and J. Frydman, 2005 Hsp110cooperates with different cytosolic HSP70 systems in a pathwayfor de novo folding. J. Biol. Chem. 280: 41252–41261.

Yamamoto, A., Y. Mizukami, and H. Sakurai, 2005 Identificationof a novel class of target genes and a novel type of bindingsequence of heat shock transcription factor in Saccharomycescerevisiae. J. Biol. Chem. 280: 11911–11919.

Yamamoto, N., Y. Maeda, A. Ikeda and H. Sakurai,2008 Regulation of thermotolerance by stress-induced tran-scription factors in Saccharomyces cerevisiae. Eukaryot. Cell.7: 783–790.

Yan, C., L. H. Lee, and L. I. Davis, 1998 Crm1p mediates regulatednuclear export of a yeast AP-1-like transcription factor. EMBO J.17: 7416–7429.

Yang, R., S. A. Wek, and R. C. Wek, 2000 Glucose limitationinduces GCN4 translation by activation of Gcn2 protein kinase.Mol. Cell. Biol. 20: 2706–2717.

Yao, J., K. M. Munson, W. W. Webb, and J. T. Lis, 2006 Dynamicsof heat shock factor association with native gene loci in livingcells. Nature 442: 1050–1053.

Young, J. C., N. J. Hoogenraad, and F. U. Hartl, 2003 Molecularchaperones Hsp90 and Hsp70 deliver preproteins to the mito-chondrial import receptor Tom70. Cell 112: 41–50.

Zarzov, P., H. Boucherie, and C. Mann, 1997 A yeast heat shocktranscription factor (Hsf1) mutant is defective in both Hsc82/Hsp82 synthesis and spindle pole body duplication. J. Cell Sci.110: 1879–1891.

Zhao, J., J. Herrera-Diaz, and D. S. Gross, 2005a Domain-widedisplacement of histones by activated heat shock factor occursindependently of Swi/Snf and is not correlated with RNA poly-merase II density. Mol. Cell. Biol. 25: 8985–8999.

Zhao, R., M. Davey, Y. C. Hsu, P. Kaplanek, A. Tong et al.,2005b Navigating the chaperone network: an integrativemap of physical and genetic interactions mediated by thehsp90 chaperone. Cell 120: 715–727.

Zou, J., Y. Guo, T. Guettouche, D. F. Smith, and R. Voellmy,1998 Repression of heat shock transcription factor HSF1 acti-vation by HSP90 (HSP90 complex) that forms a stress-sensitivecomplex with HSF1. Cell 94: 471–480.

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